KR20010101203A - Use of a MEK Inhibitor for Preventing Transplant Rejection - Google Patents

Abstract

The present invention provides a method for preventing or maintaining transplant tissue rejection of organs, cells, limbs, and tissues in a mammal, comprising administering a selective MEK inhibitor, preferably a compound of formulas (I) and (II). To provide.

Description

Use of a MEK Inhibitor for Preventing Transplant Rejection

Organ and limb transplants have become a common method for treating mammals who are victims of organ disease or loss of organ function or limbs or other trauma. Commonly implanted organs include the liver, kidneys, pancreas and lungs. Other types of transplants such as skin, bone marrow, and small intestine are also common. Amputations include larger limbs, such as fingers, toes, and arms.

Transplant tissue rejection includes both humoral and cell-mediated immune responses in mammalian patients, or delayed hypersensitivity reactions. As a result, an immunosuppressive agent is administered to the patient to inhibit or at least alleviate the rejection response. Several immunosuppressants can now be used clinically, but each involves side effects. For example, cyclosporin inhibits T-cell production of several cytokines, including IL-2 (interleukin-2), IL-3, IL-4, IL-5, IFN-δ, and possibly other lymphokines It is a cyclic peptide. Cyclosporins are widely used to prevent organ rejection in allogeneic kidney, liver, and heart transplant tissues. Cyclosporins are often used in combination with other immunosuppressive agents such as corticosteroids or azathioprine. Undesirable side effects associated with cyclosporin include addictive nephropathy, hepatocytotoxicity, severe renal failure, tremor, grandmothers, and hypertension.

Another immunosuppressant is mycophenolate mofetil, a 2-morpholinoethyl ester of mycophenolic acid that is frequently used in patients undergoing allogeneic kidney transplants. This drug is often used in combination with other immunosuppressive agents, including cyclosporin and corticosteroids. Like cyclosporin, mycophenolate mofetil can cause side effects, most notably the increased risk of developing lymphoma and other malignant tumors associated with the skin. Side effects on fetal development are also noted.

In view of the foregoing, there has been a continuing need for immunosuppressive agents that are not only useful for treating or preventing transplantation tissue rejection but also have no side effects than those involved with existing therapies. According to the invention, compounds that are MEK inhibitors are useful for preventing transplant tissue rejection in mammals. In addition, these potent immunosuppressants have fewer or no side effects. Compounds administered according to the invention are described in US Pat. No. 5,525,625 and WO 98/37881, both of which are incorporated herein by reference.

Summary of the Invention

The present invention provides a method for preventing rejection of transplanted organs, tissues, and limbs in a mammal, the method comprising administering an immunosuppressive effective amount of a selective MEK inhibitor to a patient who is or will be transplanted. . In a preferred embodiment, the MEK inhibitor administered is 2- (2-amino-3-methoxyphenyl) -4-oxo-4H- [, also known as "98059" as described in US Pat. No. 5,525,625. 1] benzopyran. In another preferred embodiment, the immunosuppressive agent administered is a phenyl amine compound of formula I or II, or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof.

In formula (I), R ₁ is hydrogen, hydroxy, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, halo, trifluoromethyl, or CN. R ₂ is hydrogen. R ₃ , R ₄ and R ₅ are hydrogen, hydroxy, halo, trifluoromethyl, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, nitro, CN and-(O or NH) _m- (CH ₂ ) is independently selected from _n -R ₉ . R ₉ is hydrogen, hydroxy, COOH or NR ₁₀ R ₁₁ ; n is 0-4; m is 0 or 1; Each R ₁₀ and R ₁₁ is independently selected from hydrogen and C ₁ -C ₈ alkyl, or ₁ selected from O, S, NH, or N- (C ₁ -C ₈ alkyl), with the nitrogen to which they are attached; 3-10 membered cyclic rings optionally containing 2, or 3 additional hetero atoms may be formed. Z is COOR ₇ , tetrazolyl, CONR ₆ R ₇ , CONHNR ₁₀ R ₁₁ , or CH ₂ OR ₇ . R ₆ and R ₇ are independently hydrogen, C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, (CO) -C ₁ -C ₈ alkyl, aryl, heteroaryl, C ₃ -C ₁₀ cycloalkyl, or C ₃ -C ₁₀ (cycloalkyl optionally containing 1, 2 or 3 hetero atoms selected from O, S, NH, or N alkyl); R ₆ and R ₇ together with the nitrogen to which they are attached form a 3-10 membered cyclic ring optionally containing 1, 2 or 3 additional hetero atoms selected from O, S, NH, or N alkyl. In formula (I), any of the above alkyl, alkenyl, aryl, heteroaryl, heterocyclic, and alkynyl groups are unsubstituted or halo, hydroxy, C ₁ -C ₆ alkoxy, amino, nitro, C ₁ -C ₄ alkylamino, di (C ₁ -C ₄ ) alkylamino, C ₃ -C ₆ cycloalkyl, phenyl, phenoxy, C ₃ -C ₅ heteroaryl, or C ₃ -C ₅ heteroaryloxy .

Preferred embodiments of formula (I) are: (a) R ₁ is hydrogen, methyl, methoxy, fluorine, chlorine, or bromine; (b) R ₂ is hydrogen; (c) R ₃ , R ₄ and R ₅ are independently hydrogen, fluorine, chlorine, bromine, iodine, methyl, methoxy, or nitro; (d) R ₁₀ and R ₁₁ are independently hydrogen or methyl; (e) Z is COOR ₇ , tetrazolyl, CONR ₆ R ₇ , CONHNR ₁₀ R ₁₁ , or CH ₂ OR ₇ ; R ₆ and R ₇ are independently hydrogen, C ₁ -C ₄ alkyl, heteroaryl, or C ₃ -C ₅ cycloalkyl optionally containing one or two hetero atoms selected from O, S, or NH; R ₆ and R ₇ together with the nitrogen to which they are attached form a 5-6 membered cyclic ring optionally containing 1 or 2 additional hetero atoms selected from O, NH, or N-alkyl; Wherein any of said alkyl or aryl groups may be unsubstituted or substituted by halo, hydroxy, methoxy, ethoxy, or heteroaryloxy (eg, synthetic intermediates 2,3,4,5,6-pentafluoro Rophenyl); (f) Z is COOR ₇ ; (g) R ₇ is H, pentafluorophenyl, or tetrazolyl; (h) R ₃ , R ₄ and R ₅ are independently H, fluorine, or chlorine; (i) R ₄ is fluorine; (j) two of R ₃ , R ₄ and R ₅ are fluorine; Or (k) has a structure which is a combination of the foregoing. In a preferred embodiment of formula (I), R ₁ is methyl, fluorine, chlorine, or bromine.

Examples of preferred embodiments include methods comprising a MEK inhibitor selected from the following formula (I) compound table.

Formula (I) Compound Table

[4-Chloro-2- (1H-tetrazol-5-yl) -phenyl- (4-iodo-2-methyl-phenyl) -amine

(4-iodo-2-methyl-phenyl)-[2- (1H-tetrazol-5-yl) -phenyl] amine

[4-Nitro-2- (1H-tetrazol-5-yl) -phenyl- (4-iodo-2-methyl-phenyl) -amine

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid

3,4,5-Trifluoro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid

5-Chloro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid

Sodium 5-chloro-2- (4-iodo-2-methyl-phenylamino) -benzoate

5-Bromo-2- (4-iodo-2-methyl-phenylamino) -benzoic acid

2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzoic acid

4-Chloro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid

2- (4-iodo-2-methyl-phenylamino) -benzoic acid

5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid

5-Iodo-2- (4-iodo-2-methyl-phenylamino) -benzoic acid

2,3,5-Trifluoro-4- (4-iodo-2-methyl-phenylamino) -benzoic acid

2- (4-Iodo-phenylamino) -5-methoxy-benzoic acid

5-Methyl-2- (4-iodo-2-methyl-phenylamino) -benzoic acid

2- (4-iodo-2-methyl-phenylamino) -4-nitro-benzoic acid

2- (4-Bromo-2-methyl-phenylamino) -4-fluoro-benzoic acid

2- (2-Bromo-4-iodo-phenylamino) -5-nitro-benzoic acid

2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-benzoic acid

5-Chloro-N- (2-hydroxyethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-methyl-benzamide

N-ethyl-4-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N, N-dimethyl-benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (1H-tetrazol-5-yl) -benzamide

5-Bromo-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N, N-dimethyl-benzamide

[5-Chloro-2- (4-iodo-2-methyl-phenylamino) -benzoylamino] -acetic acid

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-propyl-benzamide

5-Bromo-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

N, N-diethyl-4-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

4-Fluoro-N- {3- [4- (2-hydroxy-ethyl) -piperazin-1-yl] -propyl} -2- (4-iodo-2-methyl-phenylamino) -benz amides

N, N-diethyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide

N-Butyl-4-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-N, N-diethyl-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N, N-dimethyl-benzamide

5-Bromo-3,4-difluoro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

N- (2,3-dihydroxy-propyl) -3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-piperidin-1-yl-ethyl) -benzamide

3,4-Difluoro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

N- (2,3-Dihydroxy-propyl) -4-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

3,4-Difluoro-N- (3-hydroxy-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyrrolidin-1-yl-ethyl) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyridin-4-yl-ethyl) -benzamide

4-Fluoro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-N- (3-dimethylamino-propyl) -3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-2-phenylamino) -N- (2-morpholin-4-yl-ethyl) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-morpholin-4-yl-ethyl) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyrrolidin-1-yl-ethyl) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyridin-4-yl-ethyl) -benzamide

N- (3-Dimethylamino-propyl) -3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

N-benzyl-4-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (2-hydroxy-ethyl) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-morpholin-4-yl-ethyl) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-piperidin-1-yl-propyl) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-piperidin-1-yl-propyl) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-thiophen-2-yl-ethyl) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyrrolidin-1-yl-ethyl) -benzamide

2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (2-morpholin-4-yl-ethyl) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N-pyridin-4-ylmethyl-benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N-pyridin-4-ylmethyl-benzamide

2- (4-Bromo-2-ylmethyl-phenylamino) -N- (3-dimethylamino-propyl) -3,4-difluoro-benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-pyridin-4-ylmethyl-benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyridin-4-yl-ethyl) -benzamide

2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (2-pyridin-4-yl-ethyl) -benzamide

2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (3-hydroxy-propyl) -benzamide

2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (2-pyrrolidin-1-yl-ethyl) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-phenethyl-benzamide

2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (2-thiophen-2-yl-ethyl) -benzamide

2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N-pyridin-4-ylmethyl-benzamide

2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N-phenethyl-benzamide

2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (2-piperidin-1-yl-ethyl) -benzamide

5-Chloro-N- {3- [4- (2-hydroxy-ethyl) -piperazin-1-yl] -propyl} -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Fluoro-N- {3- [4- (2-hydroxy-ethyl) -piperazin-1-yl] -propyl} -2- (4-iodo-2-methyl-phenylamino) -benz amides

2- (4-iodo-2-methyl-phenylamino) -5-nitro-N-pyridin-4-yl methyl-benzamide

5-Bromo-N- {3- [4- (2-hydroxy-ethyl) -piperazin-1-yl] -propyl} -2- (4-iodo-2-methyl-phenylamino) -benz amides

5-Chloro-N- (2-diethylamino-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N- (2-piperidin-1-yl-ethyl) -benzamide

(3-hydroxy-pyrrolidin-1-yl)-[5-nitro-2- (4-iodo-2-methyl-phenylamino) -phenyl] -methanone

5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyrrolidin-1-yl-ethyl) -benzamide

5-Bromo-N- (2-diethylamino-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

N- {2- [bis- (2-hydroxy-ethyl) -amino] -ethyl} -5-chloro-2- (4-iodo-2-methyl-phenylamino) -benzamide

N- {2- [bis- (2-hydroxy-ethyl) -amino] -ethyl} -5-bromo-2- (4-iodo-2-methyl-phenylamino) -benzamide

N- {3- [4- (2-hydroxy-ethyl) -piperazin-1-yl] -propyl} -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-pyridin-4-ylmethyl-benzamide

5-Bromo-2- (4-iodo-2-ethyl-phenylamino) -N- (2-pyrrolidin-1-yl-ethyl) -benzamide

5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (2-piperidin-1-yl-ethyl) -benzamide

5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyrrolidin-1-yl-ethyl) -benzamide

5-Chloro-N- (3-dimethylamino-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

N- {2- [bis- (2-hydroxy-ethyl) -amino] -ethyl} -5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-N- (3-hydroxy-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-N- (3-diethylamino-2-hydroxy-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-piperidin-1-yl-ethyl) -benzamide

5-Bromo-N- (3-hydroxy-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (3-piperidin-1-yl-propyl) -benzamide

N- {2- [bis- (2-hydroxy-ethyl) -amino] -ethyl} -2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide

5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N- (2-morpholin-4-yl-ethyl) -benzamide

5-Chloro-N- (3-diethylamino-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-N- (2-diisopropylamino-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N- (3-piperidin-1-yl-propyl) -benzamide

2- (4-iodo-2-methyl-phenylamino) -5-nitro-N- (2-piperidin-1-yl-ethyl) -benzamide

5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (2-piperazin-1-yl-ethyl) -benzamide

N- (2-Diethylamino-ethyl) -5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-N- (3-dimethylamino-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

N- (3-hydroxy-propyl) -2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide

5-Fluoro-N- (3-hydroxy-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

N- (3-Diethylamino-propyl) -5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

N- (3-Diethylamino-propyl) -2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide

5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (2-morpholin-4-yl-ethyl) -benzamide

2- (4-iodo-2-methyl-phenylamino) -5-nitro-N- (3-piperidin-1-yl-propyl) -benzamide

[5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -phenyl]-(2 or 3-hydroxy-pyrrolidin-1-yl) -methanone

5-Bromo-N- (2-diisopropylamino-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-morpholin-4-yl-ethyl) -benzamide

5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-piperidin-1-yl-propyl) -benzamide

[5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -phenyl]-[4- (2-hydroxy-ethyl) -piperazin-1-yl) -methanone

N- (3-Diethylamino-2-hydroxy-propyl) -5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

N-cyclopropyl-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Fluoro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

N-benzyloxy-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

N-benzyloxy-5-bromo-2- (4-iodo-2-methyl-phenylamino) -benzamide

2- (4-iodo-2-methyl-phenylamino) -5-nitro-N- (4-sulfamoyl-benzyl) -benzamide

5-Bromo-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

N- (2-hydroxy-ethyl) -5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzamide

N- (2-hydroxy-ethyl) -2- (4-iodo-2-ethyl-phenylamino) -5-nitro-benzamide

2- (4-iodo-2-methyl-phenylamino) -N-methyl-5-nitro-N-phenyl-benzamide

5-Chloro-N-cyclopropyl-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-phenyl-benzamide

N-allyl-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

N-benzyloxy-5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (4-sulfamoyl-benzyl) -benzamide

N-allyl-5-chloro-2- (4-iodo-2-methyl-phenylamino) -benzamide

N-cyclopropyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide

5-Bromo-N-cyclopropyl-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-phenyl-benzamide

5-iodo-2- (4-iodo-2-methyl-phenylamino) -N- (4-sulfamoyl-benzyl) -benzamide

5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (4-sulfamoyl-benzyl) -benzamide

N-allyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide

2- (4-iodo-2-methyl-phenylamino) -5-nitro-N- (4-sulfamoyl-benzyl) -benzamide

N-allyl-5-bromo-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-benzyl) -benzamide

N-cyclopropyl-5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-phenyl-benzamide

N-benzyloxy-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide

N-cyclohexyl-5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzamide

N-allyl-5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-iodo-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-benzyl) -benzamide

2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-benzyl) -5-nitro-benzamide

5-iodo-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-phenyl-benzamide

N-cyclohexyl-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-N-cyclohexyl-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-benzyl) -benzamide

5-Bromo-N-cyclohexyl-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-benzyl) -benzamide

N-cyclohexyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide

N-benzyloxy-5-bromo-2- (4-iodo-2-methyl-phenylamino) -benzamide

N-benzyloxy-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

2- (4-iodo-2-methyl-phenylamino) -N-methyl-5-nitro-N-phenyl-benzamide

5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-phenyl-benzamide

N- (2-hydroxy-ethyl) -5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-N-cyclopropyl-2- (4-iodo-2-methyl-phenylamino) -benzamide

N-allyl-5-chloro-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-phenyl-benzamide

N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide

5-Fluoro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-N-cyclopropyl-2- (4-iodo-2-methyl-phenylamino) -benzamide

N-cyclopropyl-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (4-sulfamoyl-benzyl) -benzamide

N-cyclopropyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide

N-allyl-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

N-benzyloxy-5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzamide

N-allyl-5-bromo-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (4-sulfamoyl-benzyl) -benzamide

5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-phenyl-benzamide

N-allyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -benzyl alcohol

[5-Chloro-2- (4-iodo-2-methyl-phenylamino) -phenyl] -methanol

[2- (4-iodo-2-methyl-phenylamino) -5-nitro-phenyl] -methanol

[5-Bromo-2- (4-iodo-2-methyl-phenylamino) -phenyl] -methanol

N-allyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide.

In another preferred embodiment, the MEK inhibitor is a compound of formula II.

In formula (II), R _1a is hydrogen, hydroxy, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, halo, trifluoromethyl, or CN. R _2a is hydrogen. Each of R _3a , R _4a , and R _5a is independently hydrogen, hydroxy, halo, trifluoromethyl, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, nitro, CN, and (O or NH) _m- (CH ₂ ) _n -R _9a . R _9a is hydrogen, hydroxy, CO ₂ H or NR _10a R _11a ; n is 0-4; m is 0 or 1; Each R _10a and R _11a is independently hydrogen or C ₁ -C ₈ alkyl, or 1, 2 or 3 selected from O, S, NH, or N- (C ₁ -C ₈ alkyl) with the nitrogen to which they are attached 3- to 10-membered cyclic rings optionally containing two additional hetero atoms may be formed. R _6a is hydrogen, C ₁ -C ₈ alkyl, (CO)-(C ₁ -C ₈ alkyl), aryl, aralkyl, or C ₃ -C ₁₀ cycloalkyl. R _7a optionally represents a hetero atom selected from hydrogen, C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C ₃ -C ₁₀ (cycloalkyl or O, S, or NR _9a Containing cycloalkyl). In formula (II), any alkyl, alkenyl, aryl, heterocyclic and alkynyl groups are unsubstituted or halo, hydroxy, C ₁ -C ₆ alkoxy, amino, nitro, C ₁ -C ₄ alkylamino, di _May be substituted by (C ₁ -C ₄ ) alkylamino, C ₃ -C ₆ cycloalkyl, phenyl, phenoxy, C ₃ -C ₅ heteroaryl, or C ₃ -C ₅ heteroaryloxy; Or R _6a and R _7a together with N to which they are attached will form a 5- to 10-membered cyclic ring optionally containing 1, 2 or 3 additional hetero atoms selected from O, S, or NR _10a R _11a Can be. The invention also includes pharmaceutically acceptable salts, esters, amides or prodrugs of each of the taught compounds.

Preferred embodiments of formula (II) are: (a) R _1a is H, methyl, fluorine, or chlorine; (b) R _2a is H; R _3a , R _4a , and R _5a are each H, Cl, nitro, or F; (c) R _6a is H; (d) R _7a is methyl, ethyl, 2-propenyl, propyl, butyl, pentyl, hexyl, cyclopropylmethyl, cyclobutylmethyl, cyclopropylmethyl, or cyclopropylethyl; (e) the 4 'position is I rather than Br; (f) R _4a is para for CO-NR _6a -OR _7a at the 4 position and meta F for crosslinked nitrogen; (f) R _3a or R _5a is F; (g) at least one R _3a , R _4a , and R _5a is F; (h) R _1a is methyl or chlorine; Or (i) structures which are a combination of the foregoing.

In a more preferred embodiment the MEK inhibitor is a compound selected from the table of formula (II).

Formula (II) Compound Table

4-Fluoro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (methoxy) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (prop-2-ynyloxy) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-phenoxyethoxy) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-thienylmethoxy) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (prop-2-enyloxy) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (cyclopropylmethoxy) -benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (cyclopentoxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-furylmethoxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N-ethoxy-benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (but-2-enyloxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (cyclopropylmethoxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (1-methylprop-2-ynyloxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-phenylprop-2-ynyloxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-5-phenylpent-2-ene-4-ynyloxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (prop-2-ynyloxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (propoxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (cyclobutyloxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-thienylmethoxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-methyl-prop-2-enyloxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-phenoxyethoxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (but-2-enyloxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (but-3-ynyloxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (cyclopentyloxy) -benzamide

3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3- (2-fluorophenyl) -prop-2-ynyloxy) -benzamide

5-Bromo-3,4-difluoro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (n-propoxy) -benzamide

5-Bromo-3,4-difluoro-N- (furan-3-ylmethoxy) -2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-N- (but-2-enyloxy) -3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-N-butoxy-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-but-2-enyloxy) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-pent-2-en-4-ynyloxy) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-benzyl) -N- [5- (3-methoxy-phenyl) -3-methyl-pent-2- En-4-ynyloxy] -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (prop-2-ynyloxy) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- [3- (3-methoxy-phenyl) -prop-2-ynyloxy ] -Benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (thiophen-2-ylmethoxy) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (pyridin-3-ylmethoxy) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3- (2-fluorophenyl) -prop-2-ynyloxy) -Benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (ethoxy) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (cyclopropylmethoxy) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (isopropoxy) -benzamide

5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (but-3-ynyloxy) -benzamide

5-Chloro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N- (tetrahydro-pyran-2-yloxy) -benzamide

5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N-methoxy-benzamide

4-Bromo-2- (4-iodo-2-methyl-phenylamino) -N-phenylmethoxy-benzamide

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-phenylmethoxy-benzamide

5-Fluoro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-iodo-2- (4-iodo-2-methyl-phenylamino) -N-phenylmethoxy-benzamide

5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (tetrahydropyran-2-yloxy) -benzamide

3,4-Difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (3-phenylprop-2-ynyloxy) -benzamide

3,4-Difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (3-furylmethoxy) -benzamide

3,4-Difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (2-thienylmethoxy) -benzamide

3,4-Difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (but-3-ynyloxy) -benzamide

3,4-Difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (2-methyl-prop-2-enyloxy) -benzamide

3,4-Difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (but-2-enyloxy) -benzamide

3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (methoxy) -benzamide

3,4-Difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (ethoxy) -benzamide

3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (cyclobutoxy) -benzamide

3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (isopropoxy) -benzamide

3,4-Difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (2-phenoxyethoxy) -benzamide

3,4-Difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (cyclopropylmethoxy) -benzamide

3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (n-propoxy) -benzamide

3,4-Difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (1-methyl-prop-2-ynyloxy) -benzamide

3,4-Difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (3- (3-fluorophenyl) -prop-2-ynyloxy) -benzamide

3,4-Difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (4,4-dimethylpent-2-ynyloxy) -benzamide

3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (cyclopentoxy) -benzamide

3,4,5-Trifluoro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-3,4-difluoro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-3,4-difluoro-2- (2-fluoro-4-iodo-phenylamino) -N-hydroxy-benzamide

N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -4-nitro-benzamide

3,4,5-Trifluoro-2- (2-fluoro-4-iodo-phenylamino) -N-hydroxy-benzamide

5-Chloro-3,4-difluoro-2- (2-fluoro-4-iodo-phenylamino) -N-hydroxy-benzamide

5-Bromo-2- (2-chloro-4-iodo-phenylamino) -3,4-difluoro-N-hydroxy-benzamide

2- (2-Fluoro-4-iodo-phenylamino) -N-hydroxy-4-nitro-benzamide

2- (2-Chloro-4-iodo-phenylamino) -3,4,5-trifluoro-N-hydroxy-benzamide

5-Chloro-2- (2-chloro-4-iodo-phenylamino) -3,4-difluoro-N-hydroxy-benzamide

5-Bromo-2- (2-bromo-4-iodo-phenylamino) -3,4-difluoro-N-hydroxy-benzamide

2- (2-Chloro-4-iodo-phenylamino) -N-hydroxy-4-methyl-benzamide

2- (2-Bromo-4-iodo-phenylamino) -3,4,5-trifluoro-N-hydroxy-benzamide

2- (2-Bromo-4-iodo-phenylamino) -5-chloro-3,4-difluoro-N-hydroxy-benzamide

2- (2-Bromo-4-iodo-phenylamino) -N-hydroxy-4-nitro-benzamide

4-Fluoro-2- (2-fluoro-4-iodo-phenylamino) -N-hydroxy-benzamide

3,4-Difluoro-2- (2-fluoro-4-iodo-phenylamino) -N-hydroxy-benzamide

2- (2-Chloro-4-iodo-phenylamino) -4-fluoro-N-hydroxy-benzamide

2- (2-Chloro-4-iodo-phenylamino) -3,4-difluoro-N-hydroxy-benzamide

2- (2-Bromo-4-iodo-phenylamino) -4-fluoro-N-hydroxy-benzamide

2- (2-Bromo-4-iodo-phenylamino) -3,4-difluoro-N-hydroxy-benzamide

N-cyclopropylmethoxy-3,4,5-trifluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Chloro-N-cyclopropylmethoxy-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-Bromo-N-cyclopropylmethoxy-3,4-difluoro-2- (2-fluoro-4-iodo-phenylamino) -benzamide

N-cyclopropylmethoxy-2- (4-iodo-2-methyl-phenylamino) -4-nitro-benzamide

N-cyclopropylmethoxy-3,4,5-trifluoro-2- (2-fluoro-4-iodo-phenylamino) -benzamide

5-Chloro-N-cyclopropylmethoxy-3,4-difluoro-2- (2-fluoro-4-iodo-phenylamino) -benzamide

5-Bromo-2- (2-chloro-4-iodo-phenylamino) -N-cyclopropylmethoxy-3,4-difluoro-benzamide

N-cyclopropylmethoxy-2- (2-fluoro-4-iodo-phenylamino) -4-nitro-benzamide

2- (2-Chloro-4-iodo-phenylamino) -N-cyclopropylmethoxy-3,4,5-trifluoro-benzamide

5-Chloro-2- (2-chloro-4-iodo-phenylamino) -N-cyclopropylmethoxy-3,4-difluoro-benzamide

5-Bromo-2- (2-bromo-4-iodo-phenylamino) -N-ethoxy-3,4-difluoro-benzamide

2- (2-Chloro-4-iodo-phenylamino) -N-ethoxy-4-nitro-benzamide

2- (2-Bromo-4-iodo-phenylamino) -N-cyclopropylmethoxy-3,4,5-trifluoro-benzamide

2- (2-Bromo-4-iodo-phenylamino) -5-chloro-N-cyclopropylmethoxy-3,4-difluoro-benzamide

2- (2-Bromo-4-iodo-phenylamino) -N-cyclopropylmethoxy-4-nitro-benzamide

N-cyclopropylmethoxy-4-fluoro-2- (2-fluoro-4-iodo-phenylamino) -benzamide

N-cyclopropylmethoxy-3,4-difluoro-2- (2-fluoro-4-iodo-phenylamino) -benzamide

2- (2-Chloro-4-iodo-phenylamino) -N-cyclopropylmethoxy-4-fluoro-benzamide

2- (2-Chloro-4-iodo-phenylamino) -N-cyclopropylmethoxy-3,4-difluoro-benzamide

2- (2-Bromo-4-iodo-phenylamino) -N-cyclopropylmethoxy-4-fluoro-benzamide

2- (2-Bromo-4-iodo-phenylamino) -N-cyclopropylmethoxy-3,4-difluoro-benzamide .

In a most preferred embodiment of the invention, a compound selected from the following is administered to a patient (ie, a mammal) in an amount effective to prevent or treat transplant tissue rejection:

2- (2-chloro-4-iodophenylamino) -N-cyclopropylmethoxy-3,4-difluorobenzamide (PD184352); 2- (2-methyl-4-iodophenylamino) -N-hydroxy-4-fluorobenzamide (PD170611); 2- (2-methyl-4-iodophenylamino) -N-hydroxy-3,4-difluoro-5-bromobenzamide (PD171984); 2- (2-methyl-4-iodophenylamino) -N-cyclopropylmethoxy-3,4-difluoro-5-bromobenzamide (PD177168); 2- (2-methyl-4-iodophenylamino) -N-cyclobutylmethoxy-3,4-difluoro-5-bromobenzamide (PD180841); 2- (2-chloro-4-iodophenylamino) -N-cyclopropylmethoxy-3,4-difluoro-5-bromobenzamide (PD 184161); 2- (2-chloro-4-iodophenylamino) -N-hydroxy-3,4-difluoro-5-bromobenzamide (PD184386); 2- (2-chloro-4-iodophenylamino) -N-cyclobutylmethoxy-3,4-difluorobenzamide (PD185625); 2- (2-chloro-4-iodophenylamino) -N-hydroxy-4-fluorobenzamide (PD185848); 2- (2-methyl-4-iodophenylamino) -N-hydroxy-3,4-difluorobenzamide (PD188563); 2- (2-methyl-4-iodophenylamino) -N-cyclopropylmethoxy-3,4,5-trifluorobenzamide (PD198306); And 2- (2-chloro-4-iodophenylamino) -N-cyclopropylmethoxy-4-fluorobenzamide (PD 203311); And benzoic acid derivatives thereof. For example, the benzoic acid derivative of PD 198306 is 2- (2-methyl-4-iodophenylamino) -3,4,5-trifluorobenzoic acid.

Further preferred compounds are 2- (2-chloro-4-iodophenylamino) -5-chloro-N-cyclopropylmethoxy-3,4-difluorobenzamide (PD297189), 2- (4-ioo Dophenylamino) -N-cyclopropylmethoxy-5-chloro-3,4-difluorobenzamide (PD 297190), 2- (4-iodophenylamino) -5-chloro-3,4-di Fluorobenzoic acid (PD 296711), 2- (2-chloro-4-iodophenylamino) -5-chloro-3,4-difluorobenzoic acid (PD 296770), 5-chloro-3,4-difluoro Rho-2- (4-iodo-2-methylphenylamino) -benzoic acid (PD 296767); And 5-chloro-N-cyclopropylmethoxy-3,4-difluoro-2- (4-iodo-2-methylphenylamino) -benzamide (PD 298127).

Another preferred method according to the invention is a 2- (2-chloro-4-iodophenylamino) -N-cyclopropylmethoxy-3,4-difluoro to mammals that have received or immediately prior to transplantation. Administering an immunosuppressive agent that is robbenzamide.

Another preferred method according to the invention is to use compounds which are 2- (2-methyl-4-iodophenylamino) -N-cyclopropylmethoxy-3,4,5-trifluorobenzamide.

The invention also provides synthetic methods and synthetic intermediates.

Other features and advantages of the invention are apparent in the following detailed description, examples and claims.

The present invention relates to a method for preventing transplant tissue rejection in a mammal that has undergone an organ, tissue, cell, or limb transplant. This method involves administering an effective amount of a MEK inhibitor, ideally a phenyl amine derivative.

1 is stimulated with concanavalin A (Con A) of 2- (2-chloro-4-iodophenylamino) -N-cyclopropylmethoxy-3,4-difluorobenzamide (PD 184352). Dose response performance inhibits cellular production of interleukin-2 (IL-2) in human peripheral blood mononuclear cells.

2 shows dose response performance that inhibits cellular production of IL-2 in anti-CD3 + anti-CD28 stimulated PD-184352 human peripheral blood mononuclear cells.

3 shows dose response performance that inhibits cellular production of interferon-δ (IFN-δ) in Con-A stimulated cells of PD 184352.

4 shows the ability to inhibit human mixed lymphocyte response (MLR) as measured by uptake of tritiated thymidine (3H-TDR) of PD 184352.

5 shows the dose response performance that inhibits Con-A induced T-cell proliferation of PD 184352.

6 shows dose response performance that inhibits T-cell proliferation induced by phytohemagglutinin (PHA) of PD 184352.

7 shows that there is no toxicity in the cells of PD 184352.

8 shows the inhibitory activity of several MEK inhibitors against MLR, IFN-gamma, and IL-2, and the ability to inhibit PHA and Con A-induced proliferation of these compounds with little toxicity (MTT). Compounds tested were PD 184352;

2- (2-methyl-4-iodophenylamino) -N-hydroxy-3,4-difluoro-5-bromobenzamide (PD 171984);

2- (2-methyl-4-iodophenylamino) -N-cyclopropylmethoxy-3,4-difluoro-5-bromobenzamide (PD177168);

2- (2-chloro-4-iodophenylamino) -N-cyclopropylmethoxy-3,4-difluoro-5-bromobenzamide (PD 184161); And

2- (2-chloro-4-iodophenylamino) -N-hydroxy-3,4-difluoro-5-bromobenzamide (PD184386).

9 shows the relative IL-2 inhibitory activity of several phenyl amine compounds compared to rolipram and dexamethasone (Dex).

10 shows comparative activity of inhibiting the production of IFN-δ of several phenyl amines, rolipram, and dexamethasone.

Figure 11 shows human MLR inhibitory activity compared to dexamethasone of several phenyl amine MEK inhibitors.

12 shows the activity of inhibiting human T-cell proliferation compared to dexamethasone of several phenyl amine MEK inhibitors.

FIG. 13 shows the percentage of cell death caused in human MTT testing by several phenyl amine MEK inhibitors.

The present invention provides methods for the prevention of rejection of transplanted tissue in mammals and for the control and maintenance of the transplanted tissue. The present invention is carried out by administering an immunosuppressive effective amount of a selective MEK inhibitor to a mammal that has been or is to be transplanted to prevent or inhibit rejection of the transplanted organ, limb, cell (s), or tissue. . For example, the method is carried out by administering the phenyl amine MEK inhibitor described in WO 98/37881. These are selective MEK inhibitors, ie they inhibit MEK 1 and MEK 2 without substantially inhibiting other enzymes. This method is ideally suited for preventing and inhibiting rejection of kidney, liver, lung and limb transplant tissue.

The mammal treated in accordance with the present invention is a patient who has undergone an organ, tissue, limb, or cell transplant, or just before the transplant. Those skilled in the medical art can readily determine individual patients in need of immunosuppressive agents to prevent or inhibit the rejection of external organs, limbs, cells or tissues.

Compounds of the invention are MEK inhibitors. MEK inhibitors are compounds that show MEK inhibition when tested in the assay titled "Enzyme Assay" described from column 6, 35 of US Pat. No. 5,525,625. The entire contents of US Pat. No. 5,525,625 are incorporated herein by reference. One of the MEK inhibitors is 2- (2-amino-3-methoxyphenyl) -4-oxo-4H- [1] benzopyran. In particular, the compound exhibits activity in an assay entitled “Cascade Assay for Inhibitors of the MAP Kinase Pathway” described in column 6, 36 to column 7, line 4 of US Pat. No. 5,525,625 and / or in the column of the patent The compound is a MEK inhibitor if it shows activity in the assay entitled "In Vitro MEK Assay" described in lines 7, 4 to 27.

A. Terminology

Some of the terms used herein are defined below along with their uses used throughout this specification.

As used herein, the term “aryl” means a cyclic, bicyclic, or tricyclic aromatic ring moiety having 5 to 12 carbon atoms. Examples of typical aryl groups include phenyl, naphthyl, and fluorenyl. Aryl may be substituted with 1, 2 or 3 groups selected from fluorine, chlorine, bromine, iodine, alkyl, hydroxy, alkoxy, nitro, amino, alkylamino, or dialkylamino. Typical substituted aryl groups include 3-fluorophenyl, 3,5-dimethoxyphenyl, 4-nitronaphthyl, 2-methyl-4-chloro-7-aminofluorenyl, and the like.

The term "aryloxy" means an aryl group which is bonded via an oxygen atom, for example phenoxy, 3-bromophenoxy, naphthyloxy, and 4-methyl-1-fluorenyloxy.

"Heteroaryl" means a cyclic, bicyclic, or tricyclic aromatic ring portion containing 4 to 11 carbon atoms and 1, 2, or 3 heteroatoms selected from O, S, or N. Examples include furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, thiazolyl, oxazolyl, xanthenyl, pyridyl, indolyl, pyrimidyl, naphthyridyl, pyridyl, benzimidazolyl, And triazinyl. Heteroaryl groups may be unsubstituted or substituted with 1, 2, or 3 groups selected from fluorine, chlorine, bromine, iodine, alkyl, hydroxy, alkoxy, nitro, amino, alkylamino, or dialkylamino. Examples of substituted heteroaryl groups include chloropyranyl, methylthienyl, fluoropyridyl, amino-1,4-benzisoxazinyl, nitroisoquinolinyl, and hydroxyindolyl.

Heteroaryl groups can be bonded through oxygen to form heteroaryloxy groups such as thienyloxy, isothiazolyloxy, benzofuranyloxy, pyridyloxy, and 4-methylisoquinolinyloxy.

The term "alkyl" refers to straight and branched aliphatic groups. Typical alkyl groups include methyl, ethyl, isopropyl, t-butyl, 2,3-dimethylhexyl and 1,1-dimethylpentyl. Alkyl groups may be unsubstituted or substituted by halo, hydroxy, alkoxy, amino, alkylamino, dialkylamino, cycloalkyl, aryl, aryloxy, heteroaryl, or heteroaryloxy, these terms being defined herein As shown. Typical substituted alkyl groups include chloromethyl, 3-hydroxypropyl, 2-dimethylaminobutyl, and 2- (hydroxymethylamino) ethyl. Examples of aryl and aryloxy substituted alkyl groups are phenylmethyl, 2-phenylethyl, 3-chlorophenylmethyl, 1,1-dimethyl-3- (2-nitrophenoxy) butyl, and 3,4,5-trifluoro Ronaphthylmethyl. Examples of alkyl groups substituted by heteroaryl or heteroaryloxy groups include thienylmethyl, 2-furylethyl, 6-furyloxyoctyl, 4-methylquinolyloxymethyl, and 6-isothiazolylhexyl. Cycloalkyl substituted alkyl groups include cyclopropylmethyl, 2-cyclohexylethyl, piperidyl-2-methyl, 2- (piperidin-1-yl) -ethyl, 3- (morpholin-4-yl) propyl do.

"Alkenyl" means a straight or branched carbon chain having one or two double bonds. Examples are but-2-ethyl, 2-methyl-prop-2-enyl, 1,1-dimethyl-4-hex-4-enyl, 3-ethyl-4-methyl-pent-2-enyl, and 3- Isopropyl-pent-4-enyl. Alkenyl groups may be substituted by halo, hydroxy, alkoxy, amino, alkylamino, dialkylamino, aryl, aryloxy, heteroaryl, or heteroyloxy, for example 2-bromoethenyl, 3- Hydroxy-2-butenyl, 1-aminoethenyl, 3-phenylprop-2-enyl, 6-thiethyl-hex-2-enyl, 2-furyloxy-but-2-enyl, and 4-naph Yloxy-hex-2-enyl.

"Alkynyl" means a straight or branched carbon chain having at least one triple bond. Typical alkynyl groups include prop-2-ynyl, 2-methyl-hex-5-ynyl, 3,4-dimethyl-hex-5-ynyl, and 2-ethyl-but-3-ynyl. Alkynyl groups can be substituted, such as alkyl and alkenyl groups, for example by aryl, aryloxy, heteroaryl, or heteroaryloxy, for example 4- (2-fluorophenyl) -but-3-ynyl , 3-methyl-5-thienylpent-4-ynyl, 3-phenoxy-hex-4-ynyl, and 2-furyloxy-3-methyl-hex-4-ynyl.

Alkenyl and alkynyl groups may have one or more double or triple bonds, respectively, or may have a combination of double and triple bonds. For example, typical groups having both double and triple bonds are hex-2-en-4-ynyl, 3-methyl-5-phenylpent-2-en-4-ynyl, and 3-thienyloxy-hex- 3-ene-5-ynyl.

The term "cycloalkyl" means a non-aromatic ring or a fused ring. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, bicycloheptyl, adamantyl, and cyclohexyl. The ring may optionally contain 1, 2, or 3 hetero atoms selected from O, S, or N. Such groups include tetrahydrofuryl, tetrahydropyrrolyl, octahydrobenzofuranyl, morpholinyl, piperazinyl, pyrrolidinyl, piperidinyl, octahydroindolyl, and octahydrobenzothiofuranyl. Cycloalkyl groups can be substituted with the same substituents as alkyl and alkenyl groups, such as halo, hydroxy, aryl, and heteroaryloxy. Examples include 3-hydroxycyclohexyl, 2-aminocyclopropyl, 2-phenylpyrrolidinyl, and 3-thienylmorpholin-1-yl.

The term "maintenance" means inhibiting the tendency of a mammal to reject cells, organs, limbs, or tissues that have been transplanted into or onto the mammalian body. This method is performed by administering an amount of the selective MEK inhibitor effective to prevent or inhibit rejection.

The term "patient" means an animal, including humans. Examples of patients include humans, cows, dogs, cats, goats, sheep, horses, and pigs.

Selective MEK 1 or MEK 2 inhibitors do not substantially inhibit other enzymes such as MKK3, PKC, Cdk2A, phosphorylase kinase, EGF, and PDGF receptor kinase, and C-src, and can inhibit the MEK 1 or MEK 2 enzyme. It is a compound which inhibits. In general, selective MEK1 or MEK 2 inhibitor has an IC ₅₀ for MEK 1 or MEK 2 of at least 1/50 of the IC ₅₀ for one of the other enzyme. Preferably, the selective inhibitor has an IC ₅₀ of at least one of the enzymes at least 1/100, more preferably at least 1/500, and more preferably at least 1/1000, 1/5000 of the IC _50.

B. Administration and Formulation

The MEK inhibitor of the method may be administered to the patient as part of a pharmaceutically acceptable composition. The compositions may be administered to humans and animals, orally, rectally, parenterally (intravenously, intramuscularly, or subcutaneously), subretinal algae, intravaginally, intraperitoneally, in bladder, topically (powder, ointment, or drop) Or as an oral or nasal spray.

Compositions suitable for parenteral injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for restoration to sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or carriers include water, ethanol, polyols (propylene glycol, polyethylene glycol, greserol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil), and liquor. Organic esters used, such as ethyl octoate. Proper fluidity can be maintained, for example, using a coating such as lecithin, in the case of dispersions to maintain the required particle size, and using surfactants.

These compositions may also contain auxiliaries such as preservatives, wetting, emulsifying and adjuvant. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, for example parabens, chlorobutanol, phenol, sorbic acid and the like. It is also preferred to include isotonic agents, for example, sugars, sodium chloride and the like. Delayed absorption of the injectable pharmaceutical form can be achieved by using drugs that delay absorption, such as aluminum monostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is at least one conventional inert excipient (or vehicle), such as sodium citrate, or dicalcium phosphate, or (a) filler or extender such as starch, lactose, sucrose , Glucose, mannitol, and silicic acid, (b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic, (c) curb agents, for example Glycerol, (d) disintegrants, for example agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, sodium carbonate, (e) solution coagulation retarders, for example paraffin, (f) absorption accelerators, For example, quaternary ammonium compounds, (g) wetting agents such as cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite, and (i) lubricants such as talc, calcium stearate, Stearic acid Mixed with magnesium, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include a buffer.

Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or lactose and high molecular weight polyethylene glycols and the like.

Solid dosage forms such as tablets, sugars, capsules, pills, and granules can be prepared by coatings and shells such as enteric skin and other known in the art. They may be compositions containing a milky agent and may also release the active compound or compounds in a delayed manner in certain parts of the intestine. Encapsulation compositions that can be used are polymeric substances and waxes. The active compound may also be in micro-encapsulated form, with one or more such excipients as appropriate.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, liquid dosage forms include inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl Alcohols, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular cottonseed oil, peanut oil, corn sprout oil, olive oil, castor oil, and sesame oil, glycerol, tetrahydrofurfuryl Alcohols, polyethylene glycols, fatty acid esters of sorbitan, mixtures of these substances, and the like.

In addition to the inert diluent, the composition may also include auxiliaries such as wetting agents, emulsifiers and suspending agents, sweetening agents, flavoring agents and flavoring agents.

Suspensions are added to the active compounds, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, or Mixtures of these substances and the like.

Compositions for rectal administration are preferably suppositories, which are suitable non-irritating excipients or carriers, such as coconut butter, which dissolve in the anus or vaginal cavity to release the active compounds, which are solid at room temperature or liquid at body temperature. , Polyethylene glycol, or suppository waxes.

Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays and inhalants. The active compound is mixed with physiologically acceptable carriers under sterile conditions and may require any preservatives, buffers, or propellants. Eye preparations, eye ointments, powders and solutions are also within the scope of the present invention.

Compounds of the invention can be administered to a patient at a dosage level in the range of about 0.1 to 1000 mg per day. For average adults weighing about 70 kg, dosages in the range of 0.01 to about 100 mg per kg body weight per day are preferred. However, the specific dosage used may vary. For example, the dosage may depend on several factors, including the patient's need, the severity of the condition to be treated, and the pharmacological activity of the compound used. Determination of the optimal dosage for a particular patient is known to those skilled in the art.

The compounds of the method may be administered as pharmaceutically acceptable salts, esters, amides, or prodrugs. As used herein, the term “pharmaceutically acceptable salts, esters, amides and prodrugs” is within the scope of sound medical judgment and is suitable for contact with the patient's tissue without inappropriate toxicity, irritation, allergic reactions, etc. Carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention, where the benefit / risk ratio is reasonable and effective for their intended use, and, where appropriate, zwitterionic forms of the compounds of the present invention Refers to. The term "salt" refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds of the present invention. These salts may be prepared by themselves during the final separation and purification steps of the present invention, or may be prepared by reacting a purified compound in free base form with an appropriate organic or inorganic acid and then separating the salts thus formed. Representative salts are hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosyl Latex, citrate, malate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lacthiobionate and laurylsulfonate salts and the like. These are cations based on alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium, and the like, and include, but are not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethyl Non-toxic ammonium, quaternary ammonium, and amine cations including amines, ethylamines, and the like. (See, eg, SM Berge et al . , "Pharmaceutical Salts", J. Pharm. Sci. , 1977; 66: 1-19, which is incorporated herein by reference. .)

Examples of pharmaceutically acceptable, non-toxic esters of the compounds of the present invention include C ₁ -C ₆ alkyl esters wherein the alkyl group is straight or branched. Acceptable esters also include C ₅ -C ₇ cycloalkyl esters and aralkyl esters such as but not limited to benzyl. Preference is given to C ₁ -C ₄ alkyl esters. Esters of the compounds of the present invention can be prepared according to conventional methods.

Possible pharmaceutically acceptable of the compounds of the present invention, examples of non-toxic amides are the amides derived from ammonia, a primary C ₁ -C ₆ alkyl group is a straight or branched chain alkyl amines and secondary C ₁ -C ₆ dialkyl amine Include. In the case of secondary amines the amines may also be in the form of five or six membered heterocycles containing one nitrogen atom. Preferred are amides derived from ammonia, C ₁ -C ₃ alkyl primary amines and C ₁ -C ₂ dialkyl secondary amines. Amides of the compounds of the present invention can be prepared according to conventional methods.

The term “prodrug” refers to a compound that is rapidly modified in vivo, for example by hydrolysis in blood, to form the parent compound of the above formula. The overall description is tee. Higuchi and V. V. Stella, "Pro-drugs as Novel Delivery Systems", Vol. 14, ACS Symposium Series], and Edward Bee. Provided by Edward B. Roche, Bioversible Carriers in Drug Design , American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.

In addition, the compounds of the present invention may exist in solvated forms with unsolvated and pharmaceutically acceptable solvents such as water, ethanol and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.

Some of the compounds of the present invention may exist in various stereoisomeric forms by the presence of chiral centers. Mixtures thereof, including all stereoisomers and racemic mixtures of the compounds of the present invention, are also considered to be part of the present invention.

C. Synthesis

The following examples are intended to illustrate certain embodiments of the present invention and are not intended to limit the scope of the disclosure in any way including the claims. After the priority date of the present specification, relevant synthesis and MEK inhibition data were also published in WO 99/01421 and WO 99/01426, which are incorporated by reference.

2- (4-Bromo and 4-iodo phenylamino) -benzoic acid derivatives of formula (I) can be prepared from commercially available starting materials using synthetic methodologies known to the organic chemist. Typical synthesis is carried out by reacting 4-bromo or 4-iodoaniline with benzoic acid having a leaving group at the 2-position to yield 2- (phenylamino) -benzoic acid. This reaction is shown in Scheme 1.

In the above scheme, L is a leaving group, for example halo such as fluorine.

Reactions of aniline and benzoic acid derivatives generally involve benzoic acid in an equimolar or excess of aniline with an unreactive organic solvent such as tetrahydrofuran or toluene, such as a base such as lithium diisopropylamide, n-butyl lithium, sodium hydride, It is carried out by mixing in the presence of triethylamine, and Honey's base. The reaction is generally carried out at a temperature of about -78 ° C to about 100 ° C and is usually completed within about 2 hours to about 4 days. The product can be separated by removing the solvent, for example by reduced pressure evaporation, and can be further purified by chromatography, crystallization, or distillation if desired.

2- (phenylamino) -benzoic acid (e.g., Formula I wherein R ₇ is hydrogen) is reacted with an organic or inorganic base such as pyridine, triethylamine, calcium carbonate, or sodium hydroxide to form a pharmaceutically acceptable salt. Can be generated. The free acid can also be reacted with an alcohol of the formula HOR ₇ , wherein R ₇ is other than hydrogen, for example methyl, to produce the corresponding ester. The reaction of benzoic acid with the alcohol can be carried out in the presence of a coupling agent. Typical coupling agents include 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), 1,3-dicyclohexylcarbodiimide (DCC), bromo-tris (pyrrolidino) -Phosphonium hexafluorophosphate (PyBrOP), and (benzotriazolyloxy) tripyrrolidino phosphonium hexafluorophosphate (PyBOP). Phenylamino benzoic acid and alcohol derivatives are usually mixed in approximately equimolar amounts in an unreactive organic solvent such as dichloromethane, tetrahydrofuran, chloroform, or xylene and equimolar amounts of coupling agent are added. If desired, a base such as triethylamine or diisopropylethylamine can be added to act as an acid scavenger. The coupling reaction is generally completed in about 10 minutes to 2 hours, and the product can be easily separated by removing the reaction solvent, for example by reduced pressure evaporation, and with chromatography or acetone, diethyl ether, or ethanol The product can be purified by standard methods such as recrystallization from the same solvent.

Benzamides of the invention of formula (I) wherein Z is CONR ₆ R ₇ are readily prepared by reacting the benzoic acid with an amine of formula HNR ₆ R ₇ . The reaction is carried out by reacting approximately equimolar amounts of benzoic acid and amine in the presence of a coupling agent in an unreactive organic solvent. Typical solvents are chloroform, dichloromethane, tetrahydrofuran, benzene, toluene, and xylene. Typical coupling agents include DCC, EEDQ, PyBrOP, and PyBOP. The reaction is generally complete after about 10 minutes to about 2 hours when performed at a temperature of about 0 ° C to about 60 ° C. The product amide can be easily separated by removing the reaction solvent, for example by evaporation, and subsequent purification can be carried out by conventional methods such as chromatography, crystallization, or distillation. Hydrazide (Z = CONHNR ₁₀ R ₁₁ ) is prepared by similarly coupling benzoic acid with hydrazine of the formula H ₂ HNR ₁₀ R ₁₁ .

Benzyl alcohols of the invention, wherein Z is CH ₂ OR ₆ and R ₆ is hydrogen, are readily prepared by reducing the corresponding benzoic acid according to Scheme 2 below.

Typical reducing agents commonly used include boranes in tetrahydrofuran. The reduction is usually carried out in an unreactive organic solvent such as tetrahydrofuran and is generally complete within about 2 hours to about 24 hours when performed at a temperature of about 0 ° C to about 40 ° C.

The following detailed examples describe certain compounds provided by the present invention.

Example 1

4-Fluoro-2- (4-iodo-2-methylphenylamino) benzoic acid

2.0 in tetrahydrofuran / heptane / ethylbenzene (Aldrich) solution at −78 ° C. in a stirred solution containing 3.16 g (0.0133 mol) of 2-amino-5-iodotoluene in 5 mL of tetrahydrofuran. 10 mL (0.020 mol) of M lithium diisopropylamide was added. The resulting green suspension was vigorously stirred for 15 minutes, then 1.00 g (0.00632 mol) of a solution in 10 mL of tetrahydrofuran was added. The reaction temperature was slowly raised to room temperature and then stirred at this temperature for 2 days. The reaction mixture was concentrated. Aqueous HCl (10%) was added to the concentrate and the solution was extracted with dichloromethane. The organic phase was dried (MgSO ₄ ) and boiled in a steam bath to reduce volume and cool to room temperature. The off-white fiber was collected by vacuum filtration, washed with hexane and vacuum-oven dried (76 ° C .; ca. 10 mm Hg) to yield 1.10 g (47%) of the desired material;

mp 224-229.5 ° C;

¹ H NMR (400 MHz; DMSO): δ9.72 (s, 1H), 7.97 (dd, 1H, J = 7.0, 8.7 Hz), 7.70 (d, 1H, J = 1.5 Hz), 7.57 (dd, 1H , J = 8.4, 1.9 Hz), 7.17 (d, 1H, J = 8.2 Hz), 6.61-6.53 (m, 2H), 2.18 (s, 3H);

¹³ C NMR (100 MHz; DMSO): δ 169.87, 167.60, 165.12, 150.17, 150.05, 139.83, 138.49, 136.07, 135.31, 135.20, 135.07, 125.60, 109.32, 105.09,104.87, 99,72, 99.46, 89.43, 17.52;

¹⁹ F NMR (376 MHz; DMSO): δ-104.00 to -104.07 (m);

IR (KBr) 1670 (C = O Stretch) cm ⁻¹ ;

MS (CI) M + 1 = 372.

Analytical calculations for C ₁₄ H ₁₁ FINO ₂ :

C, 45.31; H, 2.99; N 3.77.

Found: C, 45.21; H 2.77; N, 3.64.

Example 2-30

According to the general method of Example 1, the following benzoic acid of formula (I) and salts thereof were prepared.

Example No. Compound MP ℃

2 3,4,5-trifluoro-2- (4-iodo-2-methyl-phenyla 206-210

Mino) -benzoic acid

3 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) 240.5-244.5

-Benzoic acid

4 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-259.5-262

Phenylamino) -benzoic acid

5 5-chloro-2- (2-chloro-4-iodo-phenylamino)-255-260

Benzoic acid

6 5-chloro-2- (4-iodo-2-methyl-phenylamino)-234-238

Benzoic acid

7 Sodium 5-chloro-2- (4-iodo-2-methyl-phenyla 310-320 DEC

Mino) -Benzoate

8 5-Bromo-2- (4-iodo-2-methyl-phenylamino)-239.5-240

Benzoic acid

9 2- (2-chloro-4-iodo-phenylamino) -5-nitro-289-293

Benzoic acid

10 4-fluoro-2- (3-fluoro-4-iodo-2-methyl- 233-235

Phenylamino) -benzoic acid

11 2- (4-iodo-2-methyl-phenylamino) -5-nitro-264-267

Benzoic acid

12 2- (2-Fluoro-4-iodo-phenylamino) -5-nit 256-258

Ro-benzoic acid

13 2- (4-bromo-2-methyl-phenylamino) -4-fluoro 218.5-220

-Benzoic acid

14 2- (2-Bromo-4-iodo-phenylamino) -5-nitro 285-288 DEC

-Benzoic acid

15 2- (4-Bromo-2-methyl-phenylamino) -3,4-diflu 230-234

Oro-benzoic acid

16 3-Fluoro-2- (4-iodo-2-methyl-phenylamino) 218-221

-Benzoic acid

17 3,4-difluoro-2- (4-iodo-2-methoxy-phenyl 230-233

Amino) -benzoic acid

18 4-chloro-2- (4-iodo-2-methyl-phenylamino)-245-255 DEC

Benzoic acid

19 2- (4-iodo-2-methyl-phenylamino) -benzoic acid 218-223

20 5-Fluoro-2- (4-iodo-2-methyl-phenylamino)-243-46

Benzoic acid

21 5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzoic acid 241-245

22 2,3,5-trifluoro-4- (4-iodo-2-methyl-phenylamini 218-222

No) -benzoic acid

23 4-Fluoro-2- (3-chloro-4-iodo-2-methyl-phenylamini 248-252.5

No) -benzoic acid

24 2- (4-iodo-phenylamino) -5-methoxy-benzoic acid 208-211

25 3-Chloro-2- (2-chloro-4-iodo-phenylamino) -benzoic acid 232-233

26 2-Fluoro-6- (4-iodo-2-methyl-phenylamino) -benzoic acid 179-182

27 4-Fluoro-2- (2,3-dimethyl-4-iodo-2-methyl-phenylamini 258-261

No) benzoic acid

28 5-Methyl-2- (4-iodo-2-methyl-phenylamino) -benzoic acid 209.5-211

29 2-Chloro-6- (4-iodo-2-methyl-phenylamino) -benzoic acid 171-175

30 2- (4-iodo-2-methyl-phenylamino) -4-nitro-benzoic acid 251-263

Example 31

5-Chloro-N- (2-hydroxyethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide

0.1020 g (0.2632 mmol) of 5-chloro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid, 0.1 mL (in 5 mL of 1: 1 (v / v) tetrahydrofuran-dichloromethane solution) 0.15 g (0.29 mmol) of solid PyBOP powder was added directly to a stirred solution containing 1.7 mmol) of ethanolamine, and 0.05 mL (0.29 mmol) of diisopropylethylamine. The reaction mixture was stirred at rt overnight. The solvent was removed in vacuo. The crude residue was partitioned between ether (50 mL) and 10% aqueous hydrochloric acid (50 mL). The organic phase was washed with 10% aqueous sodium hydroxide (50 mL), dried (MgSO ₄ ) and concentrated in vacuo to give a tan oil which was crystallized from hexane-ether to give 0.0831 g (73%) of greenish yellow powder; mp 120-121 ° C .;

¹ H NMR (400 MHz; CDCl ₃ ): δ9.11 (s, 1H), 7.56 (d, 1H, J = 1.4 Hz), 7.46-7.41 (m, 2H), 7.20 (dd, 1H, J = 8.9 , 2.4Hz), 7.00 (t, 2H, J = 9.6Hz), 6.55 (br t, 1H), 3.86 (t, 2H, J = 5.0Hz), 3.61 (dd, 2H, J = 10.1, 5.5Hz) , 2.23 (s, 3 H), 1.56 (br s, 1 H);

IR (KBr) 3297 (OH stretch), 1627 (C═O stretch) cm ⁻¹ ;

MS (CI) M + 1 = 431.

Analytical calcd. For C ₁₆ H ₁₆ ClIN ₂ O ₂ :

C, 44.62; H, 3. 74; N 6.50.

Found: C, 44.63; H, 3.67; N, 6.30.

Example 32-48

According to the general method of Example 31, the following benzamide was prepared by reacting the corresponding benzoic acid with the corresponding amine.

Example No. Compound MP ℃

32 4-methoxy-N- (4-methoxy-phenyl) -3-nitro-benzamide 153.5-156

33 4-Fluoro-2- (4-iodo-methyl) -phenylamino) -benzia 158

mid

34 4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-meth 102.5-104.5

Teal-benzamide

35 N-ethyl-4-fluoro-2- (4-iodo-2-methyl-phenylamino) 90-91

-Benzamide

36 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N, N- oil

Dimethyl-benzamide

37 4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-285-288 DEC

(1H-tetrazol-5-yl) -benzamide

38 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -benz 180-182

amides

39 5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N, N- 137-138

Dimethyl-benzamide

40 [5-Chloro-2- (4-iodo-2-methyl-phenylamino) -benzoyl 170-173

Amino] -acetic acid

41 4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- 69-71

Propyl-Benzamide

42 5-Bromo-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-132-133.4

Phenylamino) -benzamide

43 N, N-diethyl-4-fluoro-2- (4-iodo-2-methyl-phenyla oil

Mino) -Benzamide

44 4-Fluoro-N- {3- [4- (2-hydroxy-ethyl) -piperazine 122-124

-1-yl] -propyl} -2- (4-iodo-2-methyl-phenylamino)

-Benzamide

45 N, N-diethyl-2- (4-iodo-2-methyl-phenylamino) -5- 91-93

Nitro-benzamide

46 N-butyl-4-fluoro-2- (4-iodo-2-methyl-phenylamino)-97-99

Benzamide

47 5-Chloro-N, N-diethyl-2- (4-iodo-2-methyl-phenylamino) 118-120

-Benzamide

48 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N, N- 142.5-144

Dimethyl-benzamide

Example 49

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -benzyl alcohol

4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid (0.50 g, 1.35 mmol) was added to 6 mL (6 mmol) of the cold 1.0 M borane-tetrahydrofuran complex in tetrahydrofuran solution. Dissolved. The reaction mixture was stirred overnight at room temperature under a nitrogen atmosphere. The reaction was terminated with 80 mL of methanol. Concentration in vacuo afforded a clear tan oil which was purified by MPLC. Eluting with dichloromethane gave 0.4285 g (89%) of a white solid; mp 99-100.5 ° C .;

¹ H NMR (400 MHz; DMSO): δ 7.57 (d, 1H, J = 1.7 Hz), 7.45 (dd, 1H, J = 8.4, 1.9 Hz), 7.39 (s, 1H), 7.29 (t, 1H , J = 7.5Hz), 6.89 (d, 1H, J = 8.4Hz), 6.67-6.60 (m, 1H), 5.47 (t, 1H, J = 5.5Hz), 4.49 (d, 2H, 5.1Hz), 2.14 (s, 3 H);

IR (KBr) 3372 (OH stretch) cm ^-1 ;

MS (CI) M + 1 = 358.

Analytical calculations for C ₁₄ H ₁₃ FINO:

C, 47.08; H, 3.67; N 3.92.

Found: C, 47.17; H 3.75; N, 3.72.

Example 50-52

According to the general method of Example 49, the following benzyl alcohols were prepared.

Example No. Compound MP ℃

50 [5-Chloro-2- (4-iodo-2-methyl-phenylamino) -phenyl]-82-85

Methanol

51 [2- (4-iodo-2-methyl-phenylamino) -5-nitro-phenyl] -126.5-128.5

Methanol

52 [5-bromo-2- (4-iodo-2-methyl-phenylamino) -phenyl] -60.5-63.5

Methanol

Some compounds of the invention of formula (I) were prepared using combinatorial synthesis techniques. The general method is as follows:

To a 0.8-mL autosampler vial in a metal block was added 40 μL of a 0.5 M solution of acid in DMF and 40 μL of reagent amine (2 M solution in honey base and 1 M in amine in DMF). A 0.5 M solution of PyBrop was prepared fresh and 50 μL was added to the autosampler vial. The reaction was carried out for 24 hours.

The reaction mixture was transferred to a 2-dram vial and diluted with 2 mL of ethyl acetate. The organic layer was washed with 3 mL of distilled water and the aqueous layer was washed again with 2 mL of acetate. The combined organic layers were evaporated to dryness in an open smoke hood.

The residue was taken up in 50% acetonitrile in 2 mL of water and injected into a semi-prep reversed phase column (10 mm x 25 cm, 5 μM spherical silica, pore size 115 A derived from C-18, sample was 4.7 mL / min by linear ramp) Eluted with 100% acetonitrile for 8.5 minutes, eluting with 100% acetonitrile continued for 8 minutes). Fractions were collected by monitoring at 214 nM. The residue was dissolved in chloroform and transferred to a pre-weighed vial, evaporated and then weighed again to determine yield.

Example 53-206

Prepared by a process combining the following compounds of formula (I):

Example No. Compound MS M-H

53 5-Bromo-3,4-difluoro-N- (2-hydroxy-ethyl) -510

2- (4-iodo-2-methyl-phenylamino) -benzamide

54 N- (2,3-dihydroxy-propyl) -3,4-difluoro-2- (4- 462

Iodo-2-methyl-phenylamino) -benzamide

55 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenyl 577

Amino) -N- (2-piperidin-1-yl-ethyl) -benzamide

56 3,4-Difluoro-N- (2-hydroxy-ethyl) -2- (4-iodo-432

2-methyl-phenylamino) -benzamide

57 N- (2,3-dihydroxy-propyl) -4-fluoro-2- (4-iodo 444

2-methyl-phenylamino) -benzamide

58 3,4-Difluoro-N- (3-hydroxy-propyl) -2- (4-iodo 446

2-methyl-phenylamino) -benzamide

59 5-Bromo-3,4-difluoro-2- (4-yodo-2--2-methyl-phenyla 564

Mino) -N- (2-pyrrolidin-1-yl-ethyl) -benzamide

60 5-Bromo-3,4, -difluoro-2- (4-iodo-2-methyl-phenyla 571

Mino) -N- (2-pyridin-4-yl-ethyl) -benzamide

61 4-Fluoro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-414

Phenylamino) -benzamide

62 5-Bromo-N- (3-dimethylamino-propyl) -3,4, -difluoro 551

-2- (4-iodo-2-methyl-phenylamino) -benzamide

63 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamini 580

No) -N- (2-morpholin-4-yl-ethyl) -benzamide

64 3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N-501

(2-Morpholin-4-yl-ethyl) -benzamide

65 3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N-485

(2-pyrrolidin-1-yl) -benzamide

66 3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N-493

(2-Pyridin-4-yl-ethyl) -benzamide

67 N- (3-dimethylamino-propyl) -3,4-difluoro-2- (4- 473

Iodo-2-methyl-phenylamino) -benzamide

68 N-benzyl-4-fluoro-2- (4-iodo-2-methyl-phenylamino)-460

Benzamide

69 2- (4-bromo-2-methyl-phenylamino) -3,4-difluoro-N-384

(2-hydroxy-ethyl) -benzamide

70 4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2- 483

Morpholin-4-yl-ethyl) -benzamide

71 4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3- 495

Piperidin-1-yl-propyl) -benzamide

72 3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N-513

(3-piperidin-1-yl-propyl) -benzamide

73 4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-T 480

Offen-2-yl-ethyl) -benzamide

74 4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-P 467

Ralidin-1-yl-ethyl) -benzamide

75 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (2 453

-Morpholin-4-yl-ethyl) -benzamide

76 5-Bromo-3,4, -difluoro-2- (4-iodo-2-methyl-phenyla 557

Mino) -N-pyridin-4-ylmethyl-benzamide

77 3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N-479

Pyridin-4-ylmethyl-benzamide

78 2- (4-bromo-2-methyl-phenylamino) -N- (3-dimethylamino-425

Propyl) -3,4-difluoro-benzamide

79 4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-461

Pyridin-4-ylmethyl-benzamide

80 4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-475

Pyridin-4-yl-ethyl) -benzamide

81 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N-445

(2-Pyridin-4-yl-ethyl) -benzamide

82 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N-400

(3-hydroxy-propyl) -benzamide

83 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N-437

(2-Pyrrolidin-1-yl-ethyl) -benzamide

84 4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-phenethyl 474

-Benzamide

85 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N-450

(2-thiophen-2-yl-ethyl) -benzamide

86 2- (4-Bromo-2-methyl-phenylamino) -3,4, -difluoro-N-431

Pyridin-4-ylmethyl-benzamide

87 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N-444

Phenethyl-benzamide

88 2- (4-bromo-2-methyl-phenylamino) -3,4-difluoro-N-451

(2-piperidin-1-yl-ethyl) -benzamide

89 5-Chloro-N- {3- [4- (2-hydroxy-ethyl) -piperazin-1-yl] -557 *

Propyl} -2- (4-iodo-2-methyl-phenylamino) -benzamide

90 5-fluoro-N- {3- [4- (2-hydroxy-ethyl) -piperazin-1-yl]-541 *

Propyl} -2- (4-iodo-2-methyl-phenylamino) -benzamide

91 2- (4-iodo-2-methyl-phenylamino) -5-nitro-N-pyridine-4- 487

Methyl-benzamide

92 5-Bromo-N- {3- [4- (2-hydroxy-ethyl) -piperazin-1-yl] -601 *

Propyl} -2- (4-iodo-2-methyl-phenylamino) -benzamide

93 5-chloro-N- (2-diethylamino-ethyl) -2- (4-iodo-2-methyl-486 *

Phenylamino) -benzamide

94 5-chloro-2- (4-iodo-2-methyl-phenylamino) -N- (2-piperidine 497 *

-1-yl-ethyl) -benzamide

95 (3-hydroxy-pyrrolidin-1-yl)-[2- (4-iodo-2-methyl-phenyl 466

Amino) -5-nitro-phenyl] -methanone

96 5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyrroli 484 *

Din-1-yl-ethyl) -benzamide

97 5-Bromo-N- (2-diethylamino-ethyl) -2- (4-iodo-2-methyl 530 *

Phenylamino) -benzamide

98 N- {2- [bis- (2-hydroxy-ethyl) -amino] -ethyl} -5-chloro- 518 *

2- (4-iodo-2-methyl-phenylamino) -benzamide

99 N- {2- [bis- (2-hydroxy-ethyl) -amino] -ethyl} -5-bromo-562 *

2- (4-iodo-2-methyl-phenylamino) -benzamide

100 [5-Bromo-2- (4-iodo-2-methyl-phenylamino) -phenyl]-(3- 499

Hydroxy-pyrrolidin-1-yl) -methanone

101 2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzoic acid 501

Phenethyl ester

102 N- {3- [4- (2-hydroxy-ethyl) -piperazin-1-yl] -propyl} 568 *

-2- (4-iodo-2-methyl-phenylamino) -benzamide

103 [5-Chloro-2- (4-iodo-2-methyl-phenylamino) -phenyl]-455

(3-hydroxy-pyrrolidin-1-yl) -methanone

104 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-460

Pyridin-4-ylmethyl-benzamide

105 5-bromo-2- (4-iodo-2-methyl-phenylamino) -N- (2-528 *

Pyrrolidin-1-yl-ethyl) -benzamide

106 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (2-542 *

Piperidin-1-yl-ethyl) -benzamide

107 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-468 *

(2-Pyrrolidin-1-yl-ethyl) -benzamide

108 5-chloro-N- (3-dimethylamino-propyl) -2- (4-iodo 472 *

2-methyl-phenylamino) -benzamide

109 N- {2- [bis- (2-hydroxy-ethyl) -amino] -ethyl} -5-flu 502 *

Oro-2- (4-iodo-2-methyl-phenylamino) -benzamide

110 5-chloro-N- (3-hydroxy-propyl) -2- (4-iodo-2-methyl 445 *

-Phenylamino) -benzamide

111 5-Chloro-N- (3-diethylamino-2-hydroxy-propyl) -2- 516 *

(4-iodo-2-methyl-phenylamino) -benzamide

112 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-P 482 *

Ferridin-1-yl-ethyl) -benzamide

113 5-Bromo-N- (3-hydroxy-propyl) -2- (4-iodo-2-methyl 489 *

-Phenylamino) -benzamide

114 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (3-pipe 556 *

Ridin-1-yl-propyl) -benzamide

115 N- {2- [bis- (2-hydroxy-ethyl) -amino] -ethyl} -2- (4- 529 *

Iodo-2-methyl-phenylamino) -5-nitro-benzamide

116 5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N- (2-500 *

Morpholin-4-yl-ethyl) -benzamide

117 5-Chloro-N- (3-diethylamino-propyl) -2- (4-iodo 500 *

2-methyl-phenylamino) -benzamide

118 5-chloro-N- (2-diisopropylamino-ethyl) 514 *

-2- (4-iodo-2-methyl-phenylamino) -benzamide

119 5-chloro-2- (4-iodo-2-methyl-phenylamino) -N- (3-512 *

Piperidin-1-yl-propyl) -benzamide

120 2- (4-iodo-2-methyl-phenylamino) -5-nitro-N- (2-509 *

Piperidin-1-yl-ethyl) -benzamide

121 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (2- 544 *

Piperazin-1-yl-ethyl) -benzamide

122 N- (2-diethylamino-ethyl) -5-fluoro-2- (4-iodo 470 *

2-methyl-phenylamino) -benzamide

123 5-Bromo-N- (3-dimethylamino-propyl) -2- (4-iodo 516 *

2-methyl-phenylamino) -benzamide

124 N- (3-hydroxy-propyl) -2- (4-iodo-2-methyl-phenyl 456 *

Mino) -5-nitro-benzamide

125 5-Fluoro-N- (3-hydroxy-propyl) -2- (4-iodo-429 *

2-methyl-phenylamino) -benzamide

126 N- (3-diethylamino-propyl) -5-fluoro-2- (4-iodo 484 *

2-methyl-phenylamino) -benzamide

127 N- (3-diethylamino-propyl) -2- (4-iodo-2-methyl-511 *

Phenylamino) -5-nitro-benzamide

128 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N-544 *

(2-Morpholin-4-yl-ethyl) -benzamide

129 2- (4-iodo-2-methyl-phenylamino) -5-nitro-N-523 *

(3-piperidin-1-yl-propyl) -benzamide

130 [5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -439

Phenyl]-(3-hydroxy-pyrrolidin-1-yl) -methanone

131 5-Bromo-N- (2-diisopropylamino-ethyl) -2- (4-558 *

Iodo-2-methyl-phenylamino) -benzamide

132 5-fluoro-2- (4-iodo-2-methyl-phenylamino) -N- 484 *

(2-Morpholin-4-yl-ethyl) -benzamide

133 5-fluoro-2- (4-iodo-2-methyl-phenylamino) -N-496 *

(3-piperidin-1-yl-propyl) -benzamide

134 [5-Fluoro-2- (4-iodo-2-methyl-phenylamino)-482

Phenyl]-[4- (2-hydroxy-ethyl) -piperazine-]-

135 N- (3-diethylamino-2-hydroxy-propyl) -5-fluoro 500 *

-2- (4-iodo-2-methyl-phenylamino) -benzamide

136 [5-Chloro-2- (4-iodo-2-methyl-phenylamino) -443

Benzoylamino] -acetic acid

137 2- (4-iodo-2-methyl-phenylamino) -5-nitro-N-495 *

(2-Pyrrolidin-1-yl-ethyl) -benzamide

138 N- (3-dimethylamino-propyl) -2- (4-iodo-2-methyl-483 *

Phenylamino) -5-nitro-benzamide

139 N- (2-diisopropylamino-ethyl) -5-fluoro-2- (4- 498 *

Iodo-2-methyl-phenylamino) -benzamide

140 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -thio 490

Benzoic acid S-phenethyl ester

141 5-Chloro-2- (4-iodo-2-methyl-phenylamino) -thio 506

Benzoic acid S-phenethyl ester

142 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -thio 536

Benzoic acid S-benzyl ester

143 2- (4-iodo-2-methyl-phenylamino) -5-nitro-thio 503

Benzoic acid S-benzyl ester

144 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -thio 476

Benzoic acid S-benzyl ester

145 5-chloro-2- (4-iodo-2-methyl-phenylamino) -thio 492

Benzoic acid S-benzyl ester

146 N-cyclopropyl-5-fluoro-2- (4-iodo-2-methyl-409

Phenylamino) -benzamide

147 5-Chloro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-429

Methyl-phenylamino) -benzamide

148 5-Fluoro-N- (2-hydroxy-ethyl) -2- (4-iodo-413

2-methyl-phenylamino) -benzamide

149 N-benzyloxy-5-fluoro-2- (4-iodo-2-methyl-phenyl 475

Amino) -benzamide

150 N-benzyloxy-5-bromo-2- (4-iodo-2-methyl-phenyla 593 *

Mino) -Benzamide

151 2- (4-iodo-2-methyl-phenylamino) -5-nitro-N- (4-567

Sulfamoyl-benzyl) -benzamide

152 5-Bromo-N- (2-hydroxy-ethyl) -2- (4-iodo-2-me 473

Tyl-phenylamino) -benzamide

153 N- (2-hydroxy-ethyl) -5-iodo-2- (4-iodo-2-meth 521

Tyl-phenylamino) -benzamide

154 N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamini 440

No) -5-nitro-benzamide

155 2- (4-iodo-2-methyl-phenylamino) -N-methyl-5-nitro- 486

N-phenyl-benzamide

156 5-Chloro-N-cyclopropyl-2- (4-iodo-2-methyl-phenyl 425

Amino) -benzamide

157 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-methyl 459

-N-phenyl-benzamide

158 N-allyl-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -409

Benzamide

159 N-benzyloxy-5-iodo-2- (4-iodo-2-methyl-phenylamino) 583

-Benzamide

160 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (4-sulfa 538

Moyl-benzyl) -benzamide

161 N-allyl-5-chloro-2- (4-iodo-2-methyl-phenylamino) -425

Benzamide

162 N-cyclopropyl-2- (4-iodo-2-methyl-phenylamino) -5-436

Nitro-benzamide

163 5-Bromo-N-cyclopropyl-2- (4-iodo-2-methyl-phenyl 469

Amino) -benzamide

164 5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N-methyl-475

N-phenyl-benzamide

165 5-iodo-2- (4-iodo-2-methyl-phenylamino) -N- (4-646

Sulfamoyl-benzyl) -benzamide

166 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (4-598

Sulfamoyl-benzyl) -benzamide

167 N-allyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-436

Benzamide

168 2- (4-iodo-2-methyl-phenylamino) -5-nitro-N- (4-565

Sulfamoyl-benzyl) -benzamide

169 N-allyl-5-bromo-2- (4-iodo-2-methyl-phenylamino) -469

Benzamide

170 5-fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3- 473

Methyl-benzyl) -benzamide

171 N-cyclopropyl-5-iodo-2- (4-iodo-2-methyl-phenyl 517

Amino) -benzamide

172 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N-methyl-519

N-phenyl-benzamide

173 N-benzyloxy-2- (4-iodo-2-methyl-phenylamino) -5-nitro 502

-Benzamide

174 N-cyclohexyl-5-iodo-2- (4-iodo-2-methyl-phenylamino) 559

-Benzamide

175 N-allyl-5-iodo-2- (4-iodo-2-methyl-phenylamino) -517

Benzamide

176 5-iodo-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl 581

-Benzyl) -benzamide

177 2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-benzyl) -5- 500

Nitro-benzamide

178 5-iodo-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-567

Phenyl-benzamide

179 N-cyclohexyl-5-fluoro-2- (4-iodo-2-methyl-phenylamini 451

No) -benzamide

180 5-chloro-N-cyclohexyl-2- (4-iodo-2-methyl-phenylami 467

No) -benzamide

181 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-533

Benzyl) -benzamide

182 5-Bromo-N-cyclohexyl-2- (4-iodo-2-methyl-phenylamini 511

No) -benzamide

183 5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl 489

-Benzyl) -benzamide

184 N-cyclohexyl-2- (4-iodo-2-methyl-phenylamino) -5-nit 478

Low-benzamide

185 N-benzyloxy-5-bromo-2- (4-iodo-2-methyl-phenylamino) -538

Benzamide

186 N-benzyloxy-5-fluoro-2- (4-iodo-2-methyl-phenylamini 477

No) -benzamide

187 5-Chloro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-431

Phenylamino) -benzamide

188 5-Bromo-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-475

Phenylamino) -benzamide

189 2- (4-iodo-2-methyl-phenylamino) -N-methyl-5-nitro-488

N-phenyl-benzamide

190 5-chloro-2- (4-iodo-2-methyl-phenylamino) -N-methyl-477

N-phenyl-benzamide

191 N- (2-hydroxy-ethyl) -5-iodo-2- (4-iodo-2-methyl-523

-Phenylamino) -benzamide

192 5-Chloro-N-cyclopropyl-2- (4-iodo-2-methyl-phenyl 425

Amino) -benzamide

193 N-allyl-5-chloro-2- (4-iodo-2-methyl-phenylamino) -427

Benzamide

194 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-methyl 461

-N-phenyl-benzamide

195 N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -442

5-nitro-benzamide

196 5-Fluoro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-415

Phenylamino) -benzamide

197 5-Bromo-N-cyclopropyl-2- (4-iodo-2-methyl-phenylami 472

No) -benzamide

198 N-cyclopropyl-5-fluoro-2- (4-iodo-2-methyl-phenyla 411

Mino) -Benzamide

199 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (4-sulfa 540

Moyl-benzyl) -benzamide

200 N-cyclopropyl-2- (4-yodo-2--2-methyl-phenylamino) -5-438

Nitro-benzamide

201 N-allyl-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -411

Benzamide

202 N-benzyloxy-5-iodo-2- (4-iodo-2-methyl-phenylamino) 585

-Benzamide

203 N-allyl-5-bromo-2- (4-iodo-2-methyl-phenylamino) -472

Benzamide

204 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (4-sul 601

Pamoyl-benzyl) -benzamide

205 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N-methyl 522

-N-phenyl-benzamide

206 N-allyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro 438

-Benzamide

* M + H

Example 207

Preparation of [4-Chloro-2- (1H-tetrazol-5-yl)-(4-iodo-2-methyl-phenyl) -amine

Step a: Preparation of 5-chloro-2-fluoro-benzaldehyde

To a solution of 1-chloro-4-fluorobenzene (13.06 g, 0.1 mol) in THF (180 mL) was added dropwise LDA (2M solution in THF, 50 mL, 0.1 mol) at -78 ° C. After stirring at −78 ° C. for 1.5 h, DMF (8 mL) was added to the reaction mixture and the temperature was raised to room temperature overnight. The reaction mixture was partitioned between water and Et ₂ O. The Et ₂ O layer was dried (MgSO ₄ ) and the solvent removed in vacuo to give crude aldehyde in yield of 14.95 g (94%): ¹ H NMR (CDCl ₃ ): δ, 10.3 (s, -C ( = O) H ).

Step b : Preparation of 5-chloro-2-fluoro-benzaldehyde oxime

A solution of 5-chloro-2-fluoro-benzaldehyde (10 g, 0.0631 mol), hydroxylamine hydrochloride (6.57 g, 0.0946 mol) and pyridine (8.3 mL, 0.1010 mol) in EtOH (100 mL) was heated to 75 ° C. (oil Bath temperature) for 1 hour and the solvent was removed in vacuo to afford an oil. The oil was partitioned between water and CH ₂ Cl ₂ . The CH ₂ Cl ₂ layer was dried (MgSO ₄ ) and the solvent was removed in vacuo to afford crude aldogsim as a solid. The solid was purified by medium pressure liquid chromatography on silica. Elution with CH ₂ Cl ₂ gave 4.87 g (28%) of aldoxim as a white solid: mp 95-97 ° C .;

Analytical calculation for C ₇ H ₅ NOFCL:

C, 48.44; H, 2.90; N, 8.07.

Found: C, 48.55; H, 2.69, N, 7.90

Step c : Preparation of 5-chloro-2-fluoro-benzonitrile

A solution of 5-chloro-2-fluoro-benzaldehyde oxime (3.15 g, 0.0182 mol) in acetic anhydride (150 mL) was refluxed for 16 h. The reaction mixture was cooled to room temperature and poured into saturated aqueous NaHCO ₃ (200 mL). The mixture was extracted with Et ₂ O. The Et ₂ O layer was dried (K ₂ CO ₃ ) and the solution removed to give the product as an oily solid. The product was used for next step without further purification.

Step d : Preparation of 5- (5-chloro-2-fluoro-phenyl) -1 H-tetrazole

A mixture of 5-chloro-2-fluoro-benzonitrile (2.84 g, 0.01823 mol), butanol (15 mL), sodium azide (1.543 g, 0.0237 mol), acetic acid (1.36 mL, 0.0237 mol) was refluxed for 24 hours. I was. The reaction mixture was cooled to room temperature, additional 1.543 g sodium azide was added and the reaction mixture was refluxed for an additional 24 hours. After cooling to room temperature, Et ₂ O (100 mL) and 10% aqueous NaOH (200 mL) were added sequentially. The mixture was stirred vigorously. The aqueous layer was separated, cooled in an ice-methanol bath (-15 ° C.) and conc. Acidified to pH 1 with HCl. A gray solid precipitated out. The solid was dried in vacuo at 50 ° C. to yield 1.76 g (49%) of 5- (5-chloro-2-fluoro-phenyl) -1H-tetrazole: mp partial melt at 110 ° C., complete melt at 124 ° C. ;

¹ H (400 Mz, CDCl ₃ ): δ 8.19-8.08 (m, 1 H), 7.77-7.71 (m, 1 H), 7.61-7.52 (m, 1 H); ¹³ C (100 Mz, CDCl ₃ ): δ 159.00, 156.49, 140.88, 133.02, 132.93, 130.73, 129.23, 129.21, 129.08, 126.05, 118.96, 118.73, 114.50;

MS (CI) M + 1 = 199 (100), M = 198 (6).

Step e : Preparation of [4-Chloro-2- (1H-tetrazol-5-yl)-(4-iodo-2-methyl-phenyl) -amine

To a solution of 2-methyl-4-iodoaniline (3.52 g, 0.0151 mol) in THF (25 mL) was added dropwise LDA (2 mol solution in THF, 11.33 mL, 0.02267 mol) at -78 ° C. After stirring for 0.5 h, a solution of 1- (tetrazol-5-yl) -2-fluoro-5-chlorobenzene (1.5 g, 0.00756 mol) in THF (15 mL) was added dropwise. The reaction mixture was stirred for 16 hours to raise the temperature to room temperature. Conc. The reaction was terminated with aqueous NH ₄ Cl solution and extracted with CH ₂ Cl ₂ . The organic layer was dried (MgSO ₄ ) and the solvent removed to afford the crude product as an oil. The oil was CH ₂ Cl _2- > CH ₂ Cl ₂ : MeOH (9.7: 0.3) to give 1.5 g (48%) of the desired product:

mp 205-208 ° C; ¹ H (400Mz, DMSO): δ9.13 (s, 1H), 8.00-7.99 (s, 1H), 7.69 (s, 1H), 7.55-7.52 (m, 1H), 7.43-7.40 (m, 1H) , 7.12-7.05 (m, 1 H), 2.24 (s, 3 H); ¹³ C (100 Mz, CDCl ₃ ): δ 141.87, 139.28, 138.88, 135.47, 133.71, 131.65, 128.15, 123.69, 121.94, 116.68, 87.79, 17.22; MS (CI) M + 2 = 413 (44), M + 1 = 412 (85), M = 411 (100).

Analytical calcd. For C ₁₄ H ₁₁ N ₅ ClI.0.5H ₂ O:

C, 39.97; H, 2.87; N, 16.65

Found: C, 38.87, H, 2.77; N, 16.47.

The following tetrazol-substituted phenylamines were prepared according to the general method of Example 207.

Example 208

(4-iodo-2-methyl-phenyl)-[2- (1H-tetrazol-5-yl) -phenyl] amine, mp 231 ° C. (dec)

Example 209

[4-Nitro-2- (1H-tetrazol-5-yl)-(4-iodo-2-methyl-phenyl) -amine, mp 205-208 ° C

4-Bromo and 4-iodo phenylamino benzhydroxysamic acid derivatives of formula (II) can be prepared from commercially available starting materials using synthetic methods known to those skilled in organic chemistry. Typical synthesis is carried out by reacting 4-bromo or 4-iodoaniline with benzoic acid having a leaving group at the 2-position to give phenylamino benzoic acid, followed by reacting the benzoic acid phenylamino derivative with a hydroxylamine derivative. 3).

In the above scheme, L is a leaving group, for example halo such as fluorine, chlorine, bromine or iodine, or an activated hydroxy group such as diethylphosphate, trimethylsilyloxy, p-nitrophenoxy, or phenylsulfonoxy.

The reaction of aniline and benzoic acid derivatives generally involves the use of bases such as lithium diisopropylamide, n-butyl lithium, sodium hydride, And by mixing in the presence of sodium amide. The reaction is generally carried out at a temperature of -78 ° C to about 25 ° C and is usually completed within about 2 hours to about 4 days. The product can be separated by removing the solvent, for example by reduced pressure evaporation, and if desired can be further purified by standard methods such as chromatography, crystallization, or distillation.

Phenylamino benzoic acid is then reacted in the presence of a hydroxylamine derivative HNR _6a OR _7a with a peptide coupling agent. Hydroxylamine derivatives that can be used include methoxylamine, N-ethyl-isopropoxy amine, and tetrahydro-oxazine. Typical coupling agents include 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), 1,3-dicyclohexylcarbodiimide (DCC), bromo-tris (pyrrolidino ) -Phosphonium hexafluorophosphate (PyBrOP) and (benzotriazolyloxy) tripyrrolidino phosphonium hexafluorophosphate (PyBOP). Phenylamino benzoic acid and hydroxylamino derivatives are typically mixed in approximately equimolar amounts in an unreactive organic solvent such as dichloromethane, tetrahydrofuran, chloroform, or xylene and equimolar amounts of coupling agent are added. Bases such as triethylamine or diisopropylethylamine can be added if desired to act as an acid scavenger. The coupling reaction is generally completed after about 10 minutes to about 2 hours, the product is easily separated by removing the reaction solvent, for example by reduced pressure evaporation and the product is chromatographic or with acetone, diethyl ether, or ethanol Purification by crystallization from the same solvent.

An alternative method for preparing the compounds of the present invention involves first converting benzoic acid to a hydroxamic acid derivative and then reacting the hydroxamic acid derivative with aniline. The synthesis sequence is shown in Scheme 4.

In the above scheme, L is a leaving group. General reaction conditions for the two steps of Scheme 4 are the same as described above for Scheme 3.

Another method for preparing the compounds of the present invention involves reacting phenylamino benzhydroxamic acid with an ester former as shown in Scheme 5.

In the above scheme, L is a leaving group such as halo and base is triethylamine or diisopropylamineadi.

The synthesis of compounds of formula (Π) is described in more detail in the Examples below.

Example 1a

4-Fluoro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide

(a) Preparation of 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid

2.0 M lithium di in tetrahydrofuran / heptane / ethylbenzene (Aldrich) solution at −78 ° C. in a stirred solution containing 3.16 g (0.0133 mol) of 2-amino-5-iodotoluene in 5 mL of tetrahydrofuran. 10 mL (0.020 mol) of isopropylamide was added. The resulting green suspension was vigorously stirred for 15 minutes, after which a solution of 1.00 g (0.00632 mol) of 2,4-difluorobenzoic acid in 10 mL of tetrahydrofuran was added. The reaction temperature was slowly raised to room temperature and the mixture was stirred for 2 days at that temperature. The solvent was evaporated under reduced pressure to concentrate the reaction mixture. Aqueous HCl (10%) was added to the concentrate and the solution was extracted with dichloromethane. The organic phase was dried (MgSO ₄ ) then concentrated in a steam bath to reduce volume (10 mL) and cool to room temperature. The off white fiber formed was collected by vacuum filtration, washed with hexane and dried in vacuum-oven (76 ° C .; ca. 10 mm Hg) to give 1.10 g (47%) of the desired material; mp 224-229.5 ° C;

¹ H NMR (400 MHz, DMSO): δ9.72 (s, 1H), 7.97 (dd, 1H, J = 7.0,8.7Hz), 7.70 (d, 1H, J = 1.5Hz), 7.57 (dd, 1H , J = 8.4, 1.9 Hz, 7.17 (d, 1H, J = 8.2 Hz), 6.61-6.53 (m, 2H), 2.18 (s, 3H);

¹³ C NMR (100 MHz, DMSO): δ 169.87, 166.36 (d, J _CF = 249.4 Hz), 150.11 (d, J _CF = 11.4 Hz), 139.83, 138.49, 136.07, 135.26 (d, J _CF = 11.5 Hz ), 135.07, 125.60, 109.32, 104.98 (d, J _CF = 21.1 Hz), 99.54 (d, J _CF = 26.0 Hz), 89.43, 17.52;

¹⁹ F NMR (376 MHz, DMSO): δ-104.00 to -104.07 (m);

IR (KBr) 1670 (C = O Stretch) cm ^-1 ;

MS (CI) M + 1 = 372.

Analytical calculations for C ₁₄ H ₁₁ FINO ₂ :

C, 45.31; H, 2.99; N, 3.77.

Found: C, 45.21; H, 2.77; N, 3.64.

(b) Preparation of 4-Fluoro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide

4-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid (0.6495 g, 0.001750 mol) in 31 mL eastern blood of tetrahydrofuran-dichloromethane solution, O- (tetrahydro-2H- 1.18 g (0.00227 mol) of solid PyBOP ([benzotriazole) in a stirred solution of pyran-2-yl) -hydroxylamine (0.2590 g, 0.002211 mol), and diisopropylethylamine (0.40 mL, 0.0023 mol) Iloxy] tripyrrolidino phosphonium hexafluorophosphate, Advanced Chem Tech was added directly. The reaction mixture was stirred for 30 minutes and then concentrated in vacuo. The brown oil was treated with 10% aqueous hydrochloric acid. The suspension was extracted with ether. The organic extract was washed with 10% sodium hydroxide, then further with 10% hydrochloric acid, dried (MgSO ₄ ) and concentrated in vacuo to yield 1.0 g of light brown foam. This intermediate was dissolved in 25 mL of ethanol hydrogen chloride, and the solution was left at room temperature for 15 minutes. The reaction mixture was concentrated in vacuo to give a brown oil which was purified by flash silica chromatography. Gradient elution (from 100% dichloromethane to 0.6% methanol in dichloromethane) yielded 0.2284 g of a light brown viscous oil. Scratched with pentane-hexane and dried under high vacuum to yield 0.1541 g (23%) of off-white foam; mp 61-75 ° C .;

¹ H NMR (400 MHz, DMSO): δ 11.34 (s, 1H), 9.68 (s, 1H), 9.18 (s, 1H), 7.65 (d, 1H, J = 1.5 Hz), 7.58 (dd, 1H, J = 8.7,6.8 Hz), 7.52 (dd, 1H, J = 8.4,1.9 Hz), 7.15 (d, 1H, J = 8.4 Hz), 6.74 (dd, 1H, J = 11.8,2.4 Hz), 6.62 ( ddd, 1H, J = 8.4, 8.4, 2.7 Hz), 2.18 (s, 3H);

¹³ C NMR (100 MHz, DMSO): δ 165.91, 164.36 (d, J _CF = 247.1 Hz), 146.78, 139.18, 138.77, 132.64, 130.60 (d, J _CF = 11.5 Hz), 122.23, 112.52, 104.72 (d , J = 22.1 Hz), 100.45 (d, J _CF = 25.2 Hz), 86.77, 17.03;

¹⁹ F NMR (376 MHz, DMSO): δ-107.20 to -107.27 (m);

IR (KBr) 3307 (br, OH-H stretch), 1636 (C = O stretch) cm ^-1

MS (CI) M + 1 = 387.

Analytical calculations for C ₁₄ H ₁₂ FIN ₂ O ₂ :

C, 43.54; H, 3.13; N, 7.25.

Found: C, 43.62; H, 3.24; N, 6.98.

Example 2a

5-Bromo-3,4-difluoro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide

(a) Preparation of 5-bromo-2,3,4-trifluorobenzoic acid

Heptane / tetra in a stirred solution containing 1-bromo-2,3,4-trifluorobenzene (Aldrich, 99%; 5.30 g, 0.0249 mol) in 95 mL of anhydrous tetrahydrofuran cooled to -78 ° C. 12.5 mL of 2.0 M lithium diisopropylamide in hydrofuran / ethylbenzene solution (Aldrich) was slowly added. The mixture was stirred for 1 hour and then transferred to 700 mL of stirred saturated etheric carbon dioxide solution cooled to -78 ° C by cannula. The cold bath was removed and the reaction mixture was stirred at ambient temperature for 18 hours. Dilution (10%) aqueous hydrochloric acid (ca. 500 mL) was poured into the reaction mixture medium, and the mixture was then concentrated to a solid on a rotary evaporator. The solid product was diluted with diethyl ether (150 mL) and aq. Partition between HCl (330 mL, pH 0). The aqueous phase was extracted with the second portion (100 mL) of diethyl ether and the combined etheric extracts were washed with 5% aqueous sodium hydroxide (200 mL) and water (100 mL, pH 12). The combined alkaline aqueous extracts were acidified to pH 0 with concentrated aqueous hydrochloric acid. The resulting suspension was extracted with ether (2x200 mL). The combined organic layers were dried (MgSO ₄ ), concentrated in vacuo and subjected to high vacuum until a constant mass was obtained, yielding 5.60 g (88% yield) of off-white powder: mp 139-142.5 ° C.

¹ H NMR (400 MHz, DMSO): δ 13.97 (br s, 1 H), 8.00-7.96 (m, 1 H);

¹³ C NMR (100 MHz, DMSO): δ 162.96, 129.34, 118.47, 104.54 (d, J _CF = 22.9 Hz) ,;

¹⁹ F NMR (376 MHz, DMSO): δ-120.20 to -120.31 (m), -131.75 to -131.86 (m), -154.95 to -155.07 (m);

IR (KBr) 1696 (C═O Stretch) cm ⁻¹ ;

MS (CI) M + 1 = 255.

Analytical calcd. For C ₇₄ H ₂₁ BrF ₃ O ₂ :

C, 32.97; H, 0.79; N, 0.00; Br, 31.34; F, 22.35.

Found: C, 33.18; H, 0.64; N, 0.01; Br, 30.14; F, 22.75.

(b) Preparation of 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid

6 mL of 2.0 M lithium diisopropylamide in tetrahydrofuran / heptane / ethylbenzene (Aldrich) solution in a stirred solution containing 1.88 g (0.00791 mol) of 2-amino-2-iodotoluene in 10 mL of tetrahydrofuran. (0.012 mol) was added. After the resulting green suspension was vigorously stirred for 10 minutes, a solution of 1.00 g (0.00392 mol) of 5-bromo-2,3,4-trifluorobenzoic acid in 15 mL of tetrahydrofuran was added. The cold bath was then removed and the reaction mixture was stirred for 18 hours. The mixture was concentrated and the concentrate was treated with 100 mL dilution (10%) aqueous hydrochloric acid. The resulting suspension was extracted with ether (2 × 150 mL) and the combined organic extracts were dried (MgSO ₄ ) and concentrated in vacuo to afford an orange solid. The solid was triturated with boiling dichloromethane, cooled to ambient temperature and collected by filtration. The solid was washed with dichloromethane and dried in a vacuum oven (80 ° C.) to yield 1.39 g (76%) of greenish yellow powder; mp 259.5-262 ° C .;

¹ H NMR (400 MHz, DMSO): δ 9.03 (s, 1H), 7.99 (dd, 1H, J = 7.5,1.9 Hz), 7.57 (dd, 1H, J = 1.5 Hz), 7.42 (dd, 1H, J = 8.4, 1.9 Hz), 6.70 (dd, 1H, J = 8.4,6.0 Hz), 2.24 (s, 3H);

¹⁹ F NMR (376 MHz, DMSO): δ-123.40 to -123.47 (m); -139.00 to -139.14 (m);

IR (KBr) 1667 (C = O Stretch) cm ⁻¹ ;

MS (CI) M + 1 = 469.

Analytical calcd. For C ₁₄ H ₉ BrF ₂ INO ₂ :

C, 35.93; H, 1.94; N, 2.99; Br, 17.07; F, 8.12; I, 27.11.

Found: C, 36.15; H, 1.91; N, 2.70; Br, 16.40; F, 8.46; I, 26.05.

(c) Preparation of 5-bromo-3,4-difluoro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide

5-bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid (0.51 g, 0.0011 mol) in 20 mL of eastern blood tetrahydrofuran-dichloromethane solution, 0.6794 g (0.001306) to a stirred solution comprising O- (tetrahydro-2H-pyran-2-yl) -hydroxylamine (0.15 g, 0.0013 mol), and diisopropylethylamine (0.25 ml, 0.0014 mol) mol) of solid PyBOP (Advanced Chemtech) was added directly. The reaction mixture was stirred at 24 ° C. for 10 minutes and then concentrated in vacuo to dryness. The concentrate was suspended in 100 mL of 10% aqueous hydrochloric acid. The suspension was extracted with 125 mL of diethyl ether. The ether layer was separated, washed with 75 mL of 10% aqueous sodium hydroxide and then with 100 mL of dilute acid. The ether solution was dried (MgSO ₄ ) and concentrated in vacuo to yield 0.62 g (100%) of off-white foam. Air bubbles ca. It was dissolved in 15 mL of methanolic hydrogen chloride. After 5 minutes, the solution was concentrated in vacuo to an oil and the oil was purified by flash silica chromatography. Elution with dichloromethane: dichloromethane-methanol (99: 1) gave 0.2233 g (42%) of a yellow powder. The powder was dissolved in diethyl ether and washed with dilute hydrochloric acid. The organic phase was dried (MgSO ₄ ) and concentrated in vacuo to yield 0.200 g of bubbles. The product was triturated with pentane to give 0.1525 g of powder which was repurified by flash silica chromatography. Elution with dichloromethane gave 0.0783 g (15%) of analytical pure title compound. mp 80-90 ° C .;

¹ H NMR (400 MHz, DMSO): δ 11.53 (s, 1H), 9.38 (s, 1H), 8.82 (s, 1H), 7.70 (dd, 1H, J = 7.0,1.9 Hz), 7.53 (s, 1H), 7.37 (dd, 1H, J = 8.4, 1.9 Hz), 6.55 (dd, 1H, J = 8.2, 6.5 Hz), 2.22 (s, 3H);

¹⁹ F NMR (376 MHz, DMSO): δ-126.24 to -126.29 (m), -137.71 to -137.77 (m);

IR (KBr) 3346 (br, OH stretch), 1651 (C = O stretch) cm ⁻¹ ;

MS (CI) M + 1 = 484.

Analytical calcd. For C ₁₄ H ₁₀ BrF ₂ IN ₂ O ₂ :

C, 34.81; H, 2.09; N, 5.80.

Found: C, 34.53; H, 1.73; N, 5.52.

Examples 3a-12a in the table below were prepared according to the general method of Examples 1a and 2a.

Examples 13a-77a

Examples 13a-77a are prepared by reacting an appropriately substituted phenylamino benzoic acid (eg as shown in Scheme 1) and hydroxylamine (eg (NHR _6a ) -OR _7a ) using a combinatorial synthesis method. Was prepared. The general method is as follows:

To a 0.8-mL autosampler vial in a metal block was added 40 μL of a 0.5M solution of acid in DMF and 40 μL of hydroxylamine (2M solution in Hung's base and 1M in amine in DMF). A 0.5 M solution of PyBrOP was prepared fresh and 50 μL was added to the autosampler vial. The reaction was carried out for 24 hours.

The reaction mixture was transferred to a 2-drum vial and diluted with 2 mL of ethyl acetate. The organic layer was washed with 3 mL of distilled water and the aqueous layer was washed again with 2 mL of ethyl acetate. The combined organic layers were evaporated to dryness in an open smoke hood.

The residue was taken up in 2 mL of 50% acetonitrile in water and a semi-prep reversed phase column (10 mm x 25 cm, 5 μM spherical silica, pore size 115 A derived from C-18, sample was 100% at 4.7 mL / min by linear ramp) Elution with acetonitrile for 8.5 min. Elution with 100% acetonitrile continued for 8 min.) Fractions were collected by monitoring at 214 nM. Desired fractions were evaporated using Zymark Turbocap. The product was dissolved in chloroform and transferred to a pre-weighed vial, evaporated and then weighed again to determine yield. The structure was confirmed by mass spectroscopy.

Example 3a-77a

Example No. Compound Melting Point (° C) MS (M-H +)

3a 2- (4-bromo-2-methyl-phenylamino) -4- 56-75 DEC 523

Fluoro-N-hydroxy-benzamide

4a 5-chloro-N-hydroxy-2- (4-iodo- 65 dec

`` 2-methyl-phenylamino) -benzamide

5a 5-chloro-N-hydroxy-2- (4-iodo- 62-67

2-Methyl-phenylamino) -N-methyl-benzamide

6a 5-chloro-2- (4-iodo-2-methyl-phenyla 105-108

Mino) -N- (tetrahydropyran-2-yloxy)

Benzamide

7a 5-chloro-2- (4-iodo-2-methyl-phenyla 64-68

Mino) -N-methoxybenzamide

8a 4-fluoro-N-hydroxy-2- (4-fluoro 119-135

2-methyl-phenylamino) -benzamide

9a 4-fluoro-N-hydroxy-2- (2-methyl-fe 101-103

Nylamino) -Benzamide

10a 4-fluoro-2- (4-fluoro-2-methyl-pe 142-146

Nylamino) -N- (tetrahydropyran-2-yl

Oxy) -benzamide

11a 4-fluoro-N-hydroxy-2- (4-chloro-133.5-135

2-methyl-phenylamino) -benzamide

12a 4-fluoro-2- (4-iodo-2-methyl-phenyl 107-109.5

Amino) -N-phenylmethoxy-benzamide

13a 4-fluoro-2- (4-iodo-2-methyl-phenyl 399

Amino) -N-methoxy-benzamide

14a 3,4-difluoro-2- (4-iodo-2-methyl-417

Phenylamino) -N-methoxy-benzamide

15a 2- (4-bromo-2-methyl-phenylamino) -3, 369

4-difluoro-N-methoxy-benzamide

16a 2- (4-bromo-2-methyl-phenylamino) -N-342 *

Ethoxy-3,4-difluoro-benzamide (M-EtO)

17a 5-Bromo-N-ethoxy-3,4, -difluoro 509

-2- (4-iodo-2-methyl-phenylamino)-

Benzamide

18a 3,4-difluoro-2- (4-iodo-2-methyl-445

Phenylamino) -N-isopropoxy-benzamide

19a 2- (4-bromo-2-methyl-phenylamino) -3,4- 397

Difluoro-N-isopropoxy-benzamide

20a 4-fluoro-N- (furan-3-ylmethoxy) -2- (4-465

Iodo-2-methyl-phenylamino) -benzamide

21a 3,4-difluoro-N- (furan-3-ylmethoxy) -483

2- (4-iodo-2-methyl-phenylamino) -benz

amides

22a 2- (4-bromo-2-methyl-phenylamino) -3,4-435

Difluoro-N- (furan-3-ylmethoxy) -benz

amides

23a 5-Bromo-3,4-difluoro-N- (furan-3-561

Monomethoxy) -2- (4-iodo-2-methyl-phenyl

Amino) -benzamide

24a 5-Bromo-N- (but-2-enyloxy) -3,4- 536

Difluoro-2- (4-iodo-2-methyl-phenyl

Amino) -benzamide

25a 4-fluoro-2- (4-iodo-2-methyl-phenyl 423

Amino) -N- (prop-2-ynyloxy) -benz

amides

26a 3,4-difluoro-2- (4-iodo-2-methyl-441

Phenylamino) -N- (prop-2-ynyloxy)-

Benzamide

27a 3,4-difluoro-2- (4-iodo-2-methyl-455

Phenylamino) -N- (1-methyl-prop-2-ynyl

Oxy) -benzamide

28a 2- (4-bromo-2-methyl-phenylamino) -3,4-407

Difluoro-N- (1-methyl-prop-2-ynyljade

-Benzamide

29a N- (but-3-ynyloxy) -3,4-difluoro-455

2- (4-iodo-2-methyl-phenylamino) -benz

amides

30a 2- (4-bromo-2-methylphenylamino) -N-407

(But-3-ynyloxy) -3,4-difluoro-

Benzamide

31a 5-Bromo-N- (but-3-ynyloxy) -3,4- 533

Difluoro-2- (4-iodo-2-methyl-phenyl

Amino) -benzamide

32a 3,4-difluoro-2- (4-iodo-2-methyl-517

Phenylamino) -N- (3-phenyl-prop-2-ynyl

Oxy) -benzamide

33a 3,4-difluoro-2- (4-bromo-2-methyl-469

Phenylamino) -N- (3-phenyl-prop-2-ynyl

Oxy) -benzamide

34a 3,4-difluoro-N- [3- (3-fluoro-peh 535

Yl) -prop-2-ynyloxy] -2- (4-iodo-

2-methyl-phenylamino) -benzamide

35a 2- (4-bromo-2-methyl-phenylamino) -487

3,4-difluoro-N- [3- (3-fluoro-

Phenyl) -prop-2-ynyloxy] -benzamide

36a 3,4-difluoro-N- [3- (2-fluoro-phenyl) 535

-Prop-2-ynyloxy] -2- (4-iodo-2-methyl

-Phenylamino) -benzamide

37a 5-Bromo-3,4-difluoro-N- [3- (2-flu 613

Oro-phenyl) -prop-2-ynyloxy] -2- (4-yo

Odo-2-methyl-phenylamino) -benzamide

38a 3,4-difluoro-2- (4-iodo-2-methyl-557 *

Phenylamino) -N- (3-methyl-5-phenyl-pent-2-* (M + H)

En-4-ynyloxy) -benzamide

39a 2- (4-bromo-2-methyl-phenylamino) -3,4-510

Difluoro-N- (3-methyl-5-phenyl-pent-2-

En-4-ynyloxy) -benzamide

40a N-ethoxy-3,4-difluoro-2- (4-iodo 431

2-methyl-phenylamino) -benzamide

41a 2- (4-Bromo-2-methyl-phenylamino) -N-383

Ethoxy-3,4-difluoro-benzamide

42a 4-fluoro-2- (4-iodo-2-methyl-427

Phenylamino) -N-propoxy-benzamide

43a 3,4-difluoro-2- (4-iodo-2-methyl 445

-Phenylamino) -N-propoxy-benzamide

44a 2- (4-Bromo-2-methyl-phenylamino) -3,4 397

-Difluoro-N-propoxy-benzamide

45a 5-Bromo-3,4-difluoro-2- (4-iodo-523

2-methyl-phenylamino) -N-propoxy-benz

amides

46a 4-fluoro-2- (4-iodo-2-methyl-phenyl 427

Amino) -N-isopropoxy-benzamide

47a 3,4-difluoro-2- (4-iodo-2-methyl-445

Phenylamino) -N-isopropoxy-benzamide

48a 2- (4-bromo-2-methyl-phenylamino) -3,4- 397

Difluoro-N-isopropoxy-benzamide

49a 5-Bromo-3,4-difluoro-2- (4-iodo-523

2-methyl-phenylamino) -N-isopropoxy-benz

amides

50a N-cyclobutyloxy-3,4, -difluoro-2-457

(4-iodo-2-methyl-phenylamino) -benz

amides

51a 2- (4-bromo-2-methyl-phenylamino) -N-409

Cyclobutyloxy-3,4, -difluoro-benz

amides

52a N-cyclopentyloxy-4-fluoro-2- (4- 453

Iodo-2-methyl-phenylamino) -benzia

mid

53a N-cyclopentyloxy-3,4-difluoro-2 471

-(4-iodo-2-methyl-phenylamino) -benz

amides

54a 2- (4-bromo-2-methyl-phenylamino) -N-423

Cyclopentyloxy-3,4-difluoro-benz

amides

55a N-cyclopropylmethoxy-4-fluoro-2 439

-(4-iodo-2-methyl-phenylamino) -benz

amides

56a N-cyclopropylmethoxy-3,4-difluoro 457

-2- (4-iodo-2-methyl-phenylamino) -benz

amides

57a 2- (4-bromo-2-methyl-phenylamino) -N-cy 409

Clopropylmethoxy-3,4-difluoro-benz

amides

58a 5-Bromo-N-cyclopropylmethoxy-3,4-di 435

Fluoro-2- (4-iodo-2-methyl-phenylamino)

59a 4-fluoro-2- (4-iodo-2-methyl-phenyla 505

Mino) -N- (2-phenoxy-ethoxy) -benzamide

60a 3,4-difluoro-2- (4-iodo-2-methyl-peh 523

Nylamino) -N- (2-phenoxy-ethoxy) -benzia

mid

61a 2- (4-bromo-2-methyl-phenylamino) -3,4-475

Difluoro-N- (2-phenoxy-ethoxy) -benz

amides

62a 4-fluoro-2- (4-iodo-2-methyl-phenyl 481

Amino) -N- (thiophen-2-ylmethoxy) -benz

amides

63a 3,4-difluoro-2- (4-iodo-2-methyl-499

Phenylamino) -N- (thiophen-2-ylmethoxy)-

Benzamide

64a 2- (4-bromo-2-methyl-phenylamino) -451

3,4-difluoro-N- (thiophen-2-ylme

Methoxy) -benzamide

65a 4-fluoro-2- (4-iodo-2-methyl-phenyl 439

Amino) -N- (2-methyl-allyloxy) -benzia

mid

66a 3,4-difluoro-2- (4-iodo-2-methyl-457

Phenylamino) -N- (2-methyl-allyloxy) -bene

Zamide

67a 2- (4-bromo-2-methyl-phenylamino) -3,4-410

Difluoro-N- (2-methyl-allyloxy) -benz

amides

68a N- (but-2-enyloxy) -4-fluoro-2- (4- 439

Iodo-2-methyl-phenylamino) -benzamide

69a N- (but-2-enyloxy) -3,4-difluoro- 457

2- (4-iodo-2-methyl-phenylamino)-

Benzamide

70a 2- (4-Bromo-2-methyl-phenylamino) -N- (part 410

Tri-2-enyloxy) -3,4-difluoro-benzamide

71a 3,4-difluoro-2- (4-iodo-2-methyl-441

Phenylamino) -N- (prop-2-ynyloxy) -benz

amides

72a N- (but-3-ynyloxy) -3,4, -difluoro-455

2- (4-iodo-2-methyl-phenylamino) -benz

amides

73a 2- (4-Bromo-2-methyl-phenylamino) -N-449

(4,4-dimethyl-pent-2-ynyloxy) -3,4-

Difluoro-benzamide

74a N- (but-2-enyloxy) -3,4-difluoro 457

-2- (4-iodo-2-methyl-phenylamino) -bene

Zamide

75a 2- (4-bromo-2-methyl-phenylamino) -N-410

(But-2-enyloxy) -3,4-difluoro-

Benzamide

76a N- (3-t-butyl-propyn-2-yl) -oxy-4- 479

Fluoro-2- (4-iodo-2-methyl-phenyl

Amino) -benzamide

77a 4-fluoro-2- (4-iodo-2-methyl-phenyl 577

Amino) -N-phenylmethoxy-benzamide

Physical data for the selected compound

PD 0171984

mp 80-90 ℃

PD 0184161

mp 174-175 ° C

PD 0203311

mp 141-144 ℃

PD 0297189

mp 167-169 ℃

¹ H-NMR (400 MHz; DMSO) δ 11.70 (s, 1H), 8.59 (s, 1H), 7.55 (s, 1H), 7.43 (d, 1H, J = 6.5 Hz), 7.27 (d, 1H, J = 8.7Hz), 6.46 (m, 1H), 3.42 (d, 2H, J = 7.0Hz), 0.84 (m, 1H), 0.27 (m, 2H), 0.00 (m, 2H)

PD 0297190

mp 125.5-133 ° C

¹ H-NMR (400 MHz; DMSO) δ 11.48 (s, 1H), 8.32 (s, 1H), 7.34 (d, 1H, J = 7.5 Hz), 7.28 (d, 2H, J = 8.2 Hz), 6.48 (d, 2H, J = 7.7Hz), 3.32 (d, 2H, J = 6.8Hz), 0.81 (m, 1H), 0.28 (m, 2H), 0.00 (m, 2H)

PD 0296771

mp 266.7-268.9 ℃

¹ H-NMR (400 MHz; DMSO) δ13.85 (br s, 1H), 8.99 (s, 1H), 7.87 (dd, 1H, J = 7.9,2.1 Hz), 7.55 (d, 2H, J = 8.6Hz ), 6.82 (dd, 2H, J = 8.7,2.8 Hz)

PD 0296770

mp 293.2-296.3 ° C

¹ H-NMR (400 MHz; DMSO) δ 14.05 (br s, 1 H), 9.21 (s, 1 H), 7.93 (dd, 1 H, J = 7.8, 2.2 Hz), 7.82 (d, 1H, J = 1.9 Hz ), 7.54 (dd, 1H, J = 8.6,1.9Hz), 6.82 (dd, 1H, J = 8.6,6.7Hz)

PD 0296767

mp 249-251 ℃

¹ H-NMR (400 MHz; DMSO) δ 13.99 (br s, 1H), 9.01 (s, 1H), 7.90 (dd, 1H, J = 7.9,2.3 Hz), 7.58 (d, 1H, J = 1.6 Hz ), 7.42 (dd, 1H, J = 8.4,1.9 Hz), 6.69 (dd, 1H, J = 8.4,6.0 Hz), 2.24 (m, 3H)

PD 298127

mp 127-135 ℃

5-chloro-N-cyclopropyl methoxy-3,4-difluoro-2- [4-iodo-2-methyl phenylamino] benzamide

¹ H-NMR (400 MHz; DMSO) δ 11.64 (s, 1H), 8.28 (s, 1H), 7.38 (dd, 1H, J = 7.6, 1.7 Hz), 7.31 (d, 1H, J = 1.2 Hz) , 7.15 (dd, 1H, J = 8.5,1.7 Hz), 3.35 (d, 2H, J = 7.3 Hz), 2.01 (m, 3H), 0.83 (m, 1H), 0.28 (m, 2H), 0.01 ( m, 2H)

Biological analysis

Example 1

Inhibition of IL-2 Production Induced by Concanavalin A (Con A)

Several of these phenyl amine MEK inhibitors were evaluated in a number of assays demonstrating their usefulness for preventing graft tissue rejection in mammals. One of these assays measured the ability of test compounds to inhibit IL-2 production from T-cells (T lymphocytes) present in human peripheral blood mononuclear cells (HPBMC). In this assay, cells (HPBMC) were prepared by centrifuging test tubes of heparinized blood (usually taken from healthy volunteers) at room temperature first at 1400 rpm for 10 minutes. The intermediate phase, mostly leukocytes, was removed and added to a 50 mL centrifuge tube and diluted to a volume of 40 mL with phosphate buffered saline (PBS). Diluted PBS solution was added to 50 mL centrifuge tubes containing 7 mL of Histopague (Sigma, Sp. Gr. 1.077). The mixture was centrifuged at 2200 rpm for 20 minutes at room temperature. The middle layer containing mostly peripheral blood mononuclear cells (PBMC) was removed and added to a clean 50 mL centrifuge tube. These cells were diluted with PBS to a volume of 30 mL and centrifuged at 1000 rpm for 10 minutes at room temperature. The supernatant was removed and residual cells were washed twice with 30 mL portions of PBS. PBMCs were resuspended in medium ((Roswell Park Memorial Institute No. 1640 (RPMI-1640) from Gibco BRL, Guildsburg, MD)) and 10% fetal bovine serum (FBS) culture medium. Adjusted to 2.5 × 10 ⁶ cells / mL.

Test compounds were prepared by dissolving in dimethyl sulfoxide (DMSO) at a concentration of 30 micromoles. Additional dilutions were made in RPMI-1640, then in RPMI-1640 containing 1% DMSO, so that the final in-well concentration of DMSO was 0.25% in all wells.

Concanavalin A (Con-A) was purchased from Calbiochem (Catalog No. 234567). Stock solutions were prepared by dissolving 250 mg ConA in 10 mL sterile water (25 mg / mL).

Assays were performed by adding 50 μL of the diluted test compound to the appropriate wells of the plate. 100 μL of PBMC cell solution (2.5 × 10 ⁶ cells / mL) was added to the wells. The mixture was pre-incubated for 15 minutes at 37 ° C. in a 5% carbon dioxide incubator. For HPBMC analysis, 50 μL of Con A solution (80 μg / mL ConA in RPMI-1640) was added to the appropriate wells. For HWB analysis, 50 μL of Con A solution (800 μg / mL ConA in RPMI-1640) was added to the appropriate wells. Control wells contained medium and 50 μL of RPMI-1640. Well plates were incubated for 2 days in a 5% carbon dioxide incubator at 37 ° C. After 2 days, the plates were centrifuged at 0-4 ° C. for 5 minutes at 2200 rpm. A sample of supernatant (150 μL) was taken from each well and stored at −20 ° C. until analysis. Each sample was measured for IL-2 content by an IL-2 ELISA kit (No.D2050, purchased from R & D Systems, Minneapolis, Minnesota).

The results of the above analysis are shown in FIGS. 1 and 9. Preferred compounds for use according to the invention are 2- (2-chloro-4-iodo-phenylamino) -N-cyclopropylmethoxy-3,4-difluorobenzamide, known as PD 184352. Figure 1 shows that IL-2 was not produced by non-stimulatory cells, but was produced in large quantities in the presence of Con A. This figure shows that PD 184352 causes excellent dose dependent IL-2 production inhibition and has an IC ₅₀ value of 71 nM.

9 shows the inhibition of IL-2 production caused by several phenyl amine MEK inhibitors of Formulas I and II, known immunosuppressants, 9-fluoro-11β, 17,21-trihydroxy-16α-methylpregna Dexamethasone of steroid which is -1,4-diene-3,20-dione, and phosphodiesterase-4 inhibitor which is 4- [3- (cyclopentyloxy) -4-methoxyphenyl] -2-pyrrolidinone Shown in comparison with inrollifram.

Example 2

Inhibition of IL-2 Production Induced by Anti-CD3 & Anti-CD28

Stimulation of T-cells through direct activation of T-cell receptors is a more typical physiological T-cell activation compared to when the cells are activated by mitosis, such as Con A. T-cell receptors are complex, multiple protein receptors that are collectively included in portions of the setup protein called CD3. That is, for T cells to produce IL-2, they must also be activated by co-receptors. The most prevalent and most characteristic T-cell co-receptor is CD28. Monoclonal antibodies against CD3 and CD28 can be used together to induce release of IL-2.

Anti-CD3 is BioSource Int. It was purchased from (catalog # AHS2812). The working solution was prepared to contain 10 μg / mL of anti-CD3 in PBS. 100 μL aliquots were added to appropriate wells and incubated at 37 ° C. for 3 hours and then unbound anti-CD3 was washed with PBS. Anti-CD28 is a biosource Int. (Catalog # AH0312) and added as a solution (0.5 μg / mL) to the appropriate wells after addition of inhibitors of HPBMC and MEK.

HPBMC was prepared as described in Example 1 and IL-2 was released by stimulating at a concentration of anti-CD3 and anti-CD28 determined from preliminary studies to provide high T cell activation. After a two-day incubation period in a humidified 37 ° C. incubator containing 5% CO ₂ in air, the supernatant was collected and analyzed for IL-2 as described in Example 1.

The results of the analysis are shown in FIG. Preferred compounds for use according to the invention are PD 184352. This figure shows that IL-2 was not produced by unstimulated cells, but was produced in large quantities in the presence of anti-3 and anti-CD28. This figure shows that PD 184352 causes excellent inhibition of production of dose dependent IL-2 and has an IC ₅₀ of ₄₇ nM.

Example 3

Inhibition of Interferon-gamma Production

According to the method, the ability of the phenyl amine MEK inhibitors of Formulas I and II to inhibit the release of interferon gamma (IFN-gamma) from human peripheral blood mononuclear cells (HPBMC) and human whole blood (HWB) was evaluated. Cell samples and compound samples were prepared according to the general method described above. Analysis of cultured well plates was performed using the IFN-gamma ELISA kit (No.DIF00 from R & D Systems). The analysis results are shown in FIGS. 3 and 10. 3 shows that Con A causes the mass production of IFN-gamma and that production is completely inhibited at certain concentrations by PD 184352. The figure shows that the IC ₅₀ of PD 184352 for IFN-gamma is 148 nM.

FIG. 10 shows the dose dependent inhibition of IFN-gamma caused by several phenyl amine MEK inhibitors of Formulas I and II and the activity of known immunosuppressive agents rolipram and dexamethasone. This data demonstrates that phenyl amine MEK inhibitors are more effective than rolipram and cause almost 100% inhibition at concentrations above 1 μM. The ability to inhibit IFN-gamma production of phenyl amine MEK inhibitors of Formulas (I) and (II) demonstrates that they can be used to prevent rejection of transplanted tissue of organs, limbs, cells and tissues in mammals.

Example 4

Human Mixed Lymphocyte Reaction

Lymphocytes (or leukocytes) from donors with several MEK inhibitors (eg, potential recipients of transplanted tissue) used in the methods of the present invention may be transferred to other donors (eg, potential transplanted tissue donors, generally living cognates). Assessed in an in vitro test incubated in the presence of leukocytes from a donor, not a cadaveric donor). This test measured the degree of transplant rejection. The assay is a mixed lymphocyte (or leukocyte) response and is referred to as "MRL". In this assay, inhibition of tritiated thymidine ( ³ H-TDR) incorporation was measured. Tritiated thymidine was supplied by Amersham (Catalog No. TRK.758, 250 μCi). The commercial product was diluted in RPMI-1640 in a 50 mL conical centrifuge tube to give a working stock solution of 5-10 μCi / mL. Cells and test compounds were prepared as described above. Compounds and cells were incubated at 37 ° C. in a 5% carbon dioxide incubator. On day 6, each well of the assay plate was labeled with ³ H-TDR working stock solution (total 0.1-0.5 μCi per well). Plates were labeled and incubated for an additional 6 hours. Plate samples were recovered using a multichannel harvester and the radioactivity of each sample was counted using a betaplate Wallace 1205 counter.

4 shows the activity of PD 184352 in human MLR assay. Activity was measured as count per minute (CPM) of tritiated thymidine ( ³ H-TDR) uptake. The figure shows a dose dependent inhibition of ³ H-TDR uptake, with almost complete inhibition at 10 μM, while the untreated MLR values exceeded 4500 CPM. The IC ₅₀ for PD 184352 proved to be 186 nM.

FIG. 11 shows the activity in MLR analysis of several phenyl amine MEK inhibitors in comparison to dexamethasone.

The data shown in FIGS. 4 and 11 further demonstrated that the selective MEK inhibitors of Formulas I and II are useful for preventing rejection of transplanted organs, tissues, cells and limbs in mammals.

Example 5

Inhibition of T-cell proliferation induced by Con A

Another measure of immunosuppressive activity is the ability of compounds to block the growth of T-cells. Non-suppressive proliferation of T-cells causes rejection of organs, tissues, cells, and limbs implanted in mammals. Immunological studies have demonstrated that cyclosporin A blocks the activation of T cells, which is in part a result of inhibition of the synthesis of interleukin-2, a major growth factor for T-cells. The assay was performed by preparing cells and test compounds according to the general method described above and measuring ³ H-TDR inhibition. Con A was used to induce T-cell proliferation.

5 shows the extent to which PD 184352 inhibits T-cell proliferation. That is, the compound caused about 50% inhibition of Con A induced proliferation at the lowest test dose (0.12 μM) and nearly complete inhibition at the highest test dose (10.0 μM). The IC ₅₀ for the compound was determined to be 340 nM.

FIG. 12 shows that all of the phenyl amine MEK inhibitors tested resulted in inhibition of T-cell proliferation that is excellent and dose dependent.

Example 6

Inhibition of T-cell proliferation induced by plant hemagglutinin (PHA)

T-cell inhibition studies were performed using reagent PHA to induce proliferation. 6 shows the effect of PD 184352. In this study, test compounds failed to cause inhibition at the lowest dose (0.12 μM), but resulted in measurable inhibition at all other doses, and almost complete inhibition at the highest dose (10 μM). In this analysis, the IC ₅₀ was determined to be 1.9 μM. This data further demonstrates the ability of phenyl amine MEK inhibitors to inhibit T-cell proliferation and thereby useful in the prevention of transplant tissue rejection in mammals.

Example 7

Toxicity Analysis

As mentioned above, the MEK inhibitors used in the methods of the present invention are potent inhibitors of graft tissue rejection and at the same time have little toxicity, a property that greatly limits the clinical utility of commercial immunosuppressants. The toxic effects of the compounds were evaluated in the assay using MTT, which is a chemical known as 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide. MTT changes color when activated by cells, and the color change can be measured by routine methods. Only living cells can change the color of MTT. For this analysis, live U-937 cells were obtained from the American Type Culture Collection (Rockville, Maryland). PD 184352 was added to the cells in the wells and the cells were cultured as described above. After the incubation period, the color change of MTT was measured using a spectrophotometer. FIG. 7 shows that PD 184352 did not cause toxicity at concentrations below 33 μM and only caused slight color changes at high concentrations of 100 μM. The dose of PD 184352 (TC ₅₀ ) required to kill half of the cells was determined to be at least 100 μΜ. These data demonstrate that the phenyl amine MEK inhibitor showed virtually no any toxic effect in this assay.

13 shows the toxicity of some phenyl amine MEK inhibitors evaluated in MTT assay. The data demonstrate that the therapeutic index of all compounds evaluated, ie the biological efficacy of preventing graft tissue rejection against toxicity, is very beneficial. Therefore, there will be widespread use of this compound in clinical settings for preventing and inhibiting graft tissue rejection in mammals.

FIG. 8 shows comparative activity in a number of the assays described above, compared to the activities of rolipram and dexamethasone of several phenyl amine MEK inhibitors of Formulas I and II. The figure demonstrates that phenyl amine MEK inhibitors generally have activity beyond known drug as assessed by standard assays demonstrating the usefulness of the compounds for the prevention of graft tissue rejection in mammals.

Many of these data are summarized in Pharmacological Table 1. The table shows the in vitro effects on human leukocytes of some compounds used in the present invention and some comparative immunosuppressive agents. The data is the concentration of test compound (IC ₅₀ ) required to induce 50% inhibition of the measured parameters, with the exception of the toxicity data indicated as TC ₅₀ (the concentration required to induce toxicity in 50% of cells). In the table, "APK" refers to the activity of a compound in the cascade assay, where the compound inhibits the MEK enzyme, thereby inhibiting phosphorylation of another enzyme, MAP (mitogen activating protein) kinase, Otherwise it results in phosphorylation of the substrate, which in this assay is the myelin basic protein. Comparative Medication U0126 (in pharmacological Table 1) is 1,4-diamino-2,3-dicyano-1,4-bis [2-amino, an immunosuppressive compound described in US Pat. No. 2,779,780. Phenylthio] butadiene.

Pharmacological Table 1 MEK inhibitors: In vitro effects on human leukocytes (except TC ₅₀ phosphorylation data (MTT), all data refer to (*) IC ₅₀ or% inhibition at a given concentration) APKIC50 (nM) Human IL-2 (μM) IFN-gamma (μM) U937MTTTC ₅₀ (μM) or (% kill) ³ H-TDRPHA (μM) ³ H-TDRMLR (μM) ³ H-TDRcon A (μM) 171984-0000177098-0000177168-0000 3.014.018.0 0.0190.0060.052 * 0.034 * 0.076 * 0.17 50.6 * 29.9 * 13.9 * 2.4 * NJ * 2.5 0.35 * NJ0.69 * 0.19 * 3.9 * 0.52 180841-0000184161-0000] 84352-0000184386-0000185625-0000 4.41.61.31.45.1 * O.11 * 0.190.0680.0390.071 * 0.21 * 0.15 * 0.14 * 0.040 * 0.12 * 5.8 * 12.6> 100 (6%) 61 * 12.4 * ND * 1.8 * 4.5 * 4.0 * NJ * NJ0.530.640.41 * 0.39 * ND * 0.61 * 0.52 * 0.31 * 4.0 185848-0000188563-0000198306-0000203311-0000 1.01.38.0-- 0.0180.0130.0370.032 * 0.024 * 0.15 * 0.15 * 0.10 38.14013.1 * 33.2 * NJ * NJ * 1.40 * ND * 0.51 * 0.17 * 0.18 * 0.076 * NJ * NJ * 1.9 * ND Standard 98059-0000U0126 (PD 199601) Ralipramdexamemethasone > 1000 ------ * 7.40.0770.094 * 0.005 * 5.8 * 0.25 * 0.65 * 0.005 > 100 (0%)> 100 (0%) * ND> 100 (0%) *> 10 * NJ *> 10 *> 10 * 5.1 * 0.83 * 3.5 * 0.01 *> 10 * NJ * NJ * <0.041 ND = No measurement. NJ = No judgment: The study is not measurable or incomplete.

This extensive biological evaluation clearly demonstrates that the selective MEK inhibitors described above, in particular the phenyl amines of formulas (I) and (II), are very suitable for preventing transplantation tissue rejection in mammals, preferably humans. As with other immunosuppressants, MEK inhibitors can be used in combination with other immunosuppressants for better results. For example, MEK inhibitors can be clinically combined with other known immunosuppressive agents such as cyclosporin A and FK 506. These drugs may be combined in the same formulation, but more typically in their individual formulation dosages, and usually at the dosage levels routinely used for the individual reagents when used alone; However, lower or higher doses may be used if desired. The individual medications may be packaged together in a kit or the like for the convenience of the physician.

Example 8

Selective MEK inhibitors used in the methods of the present invention will be further evaluated in in vivo assays demonstrating their ability to prevent and inhibit graft tissue rejection. A typical in vivo assay is a homogeneous mouse ear-heart model using neonatal mouse hearts. Mice of the BL / 6 to C3H species will be used as test animals. Ten mice will be treated with MEK inhibitors. Three carrier control heterozygous grafts were included and three control factor grafts were also included as control animals. Mice will be administered twice daily at 50 mg / kg until the tissue is rejected or until there is clear evidence of anti-rejection. The MEK inhibitors evaluated will be dissolved in a dosing solution of 10% ethanol, 10% Cremophor EL (Sigma, Cat. No. C-5135) and 80% water (v / v / v). Administration is administered orally to test animals using tuberculin syringes and mouse oral nutrient tubes. The dose rate is 0.1 mL solution per 20 g of mouse body weight. The MEK inhibitor tested (300 mg) is placed in a 50 mL conical tube and 3.0 mL of ethanol is added. Block the tube to delay evaporation and vortex to promote dissolution. Cremophor EL (3.0 mL) was added followed by 24.0 mL of water. 30 mL of the dosing solution was vortexed and stored until used at 5 ° C.

When any transplant is rejected at any time during the study, the animal is sacrificed by dry ice (CO ₂ ) choking as soon as the rejection is measured for rejection. All samples are taken immediately after sacrifice and placed in 10-20 mL buffered formalin. If all bifactorial transplants survive to the end of the study, half are placed in buffered formalin and the other half frozen for later analysis. The following tissues are collected for histopathology and phospho-ERK analysis: ears including two factor grafts (or driver factor grafts); Ipsilateral cervical lymph node; Contralateral cervical lymph nodes; spleen; And heparinized blood collected by cardiac puncture for drug concentration determination. If the transplanted tissue survives by day 50, the study is terminated and the specimen collected and analyzed.

The methods of the present invention provide for both the prevention and maintenance of a patient who receives or will receive a transplant. Evaluation of the MEK inhibitor, 2- (2-methyl-4-iodophenylamino) -N-cyclopropylmethoxy-3,4,5-trifluorobenzamide (PD 198306) was performed using this protocol. , No improvement in survival of transplanted tissue was observed (data not shown). This may be due to any or several combinations of factors, inter alia due to insufficient exposure of the target cells to appropriate and sustained concentrations for MEK inhibition. Survival time for cofactorial transplantation in mice treated with PD 198306 was somewhat shortened, suggesting that MEK inhibitors may be more effective against transplantation tissue maintenance.

D. Other Embodiments

From the foregoing description and examples, and from the claims below, the essential features of the invention will be apparent. The scope of the present invention also includes various modifications within the knowledge of those skilled in the art. Examples include taught compounds or esters, pharmaceutical salts, hydrates, acids, or amides of the taught compounds modified by the addition or removal of protecting groups. The documents cited herein are incorporated by reference in their entirety.

Claims (15)

A method for preventing and inhibiting rejection of a transplanted organ, cell, tissue, or limb of a patient, comprising administering an immunosuppressive effective amount of a MEK inhibitor to a patient who has received or will receive a transplant. The method of claim 1, wherein the MEK inhibitor administered is 2- (2-amino-3-methoxyphenyl) -4-oxo-4H- [1] benzopyran. The method of claim 1, wherein the MEK inhibitor is a selective MEK 1 or MEK 2 inhibitor. The method of claim 1, wherein the MEK inhibitor is a compound of Formula I: or a pharmaceutically acceptable salt, ester, amide or prodrug thereof. <Formula I> Wherein R ₁ is hydrogen, hydroxy, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, halo, trifluoromethyl, or CN; R ₂ is hydrogen; R ₃ , R ₄ and R ₅ are independently hydrogen, hydroxy, halo, trifluoromethyl, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, nitro, CN or-(O or NH) _m- ( CH ₂ ) _n -R ₉ , wherein R ₉ is hydrogen, hydroxy, COOH or NR ₁₀ R ₁₁ ; n is 0-4; m is 0 or 1; R ₁₀ and R ₁₁ are independently hydrogen or C ₁ -C ₈ alkyl, or 1, 2, or 3 additional groups selected from O, S, NH, or NC ₁ -C ₈ alkyl with the nitrogen to which they are attached; To form a 3-10 membered cyclic ring optionally containing a hetero atom; Z is COOR ₇ , tetrazolyl, CONR ₆ R ₇ , CONHNR ₁₀ R ₁₁ , or CH ₂ OR ₇ ; R ₆ and R ₇ are independently hydrogen, C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, (CO) -C ₁ -C ₈ alkyl, aryl, heteroaryl, C ₃ -C ₁₀ cycloalkyl, or C ₃ -C ₁₀ (cycloalkyl optionally containing 1, 2 or 3 hetero atoms selected from O, S, NH, or N alkyl); R ₆ and R ₇ together with the nitrogen to which they are attached form a 3-10 membered cyclic ring optionally containing 1, 2 or 3 additional hetero atoms selected from O, S, NH, or N alkyl; Wherein said alkyl, alkenyl, aryl, heterocyclic, and alkynyl groups are unsubstituted or halo, hydroxy, C ₁ -C ₆ alkoxy, amino, nitro, C ₁ -C ₄ alkylamino, di (C ₁ -C ₄ ) alkylamino, C ₃ -C ₆ cycloalkyl, phenyl, phenoxy, C ₃ -C ₅ heteroaryl, or C ₃ -C ₅ heteroaryloxy. The method according to claim 4, wherein the MEK inhibitor, [4-Chloro-2- (1H-tetrazol-5-yl) -phenyl- (4-iodo-2-methyl-phenyl) -amine; (4-iodo-2-methyl-phenyl)-[2- (1H-tetrazol-5-yl) -phenyl] amine; [4-nitro-2- (1H-tetrazol-5-yl) -phenyl- (4-iodo-2-methyl-phenyl) -amine; 4-fluoro-2- (4-iodo-2-methylphenylamino) benzoic acid; 3,4,5-trifluoro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid; 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid; 5-Chloro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid; Sodium 5-chloro-2- (4-iodo-2-methyl-phenylamino) -benzoate; 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -benzoic acid; 2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzoic acid; 4-Chloro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid; 2- (4-iodo-2-methyl-phenylamino) -benzoic acid; 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -benzoic acid; 5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzoic acid; 2,3,5-trifluoro-4- (4-iodo-2-methyl-phenylamino) -benzoic acid; 2- (4-iodo-phenylamino) -5-methoxy-benzoic acid; 5-Methyl-2- (4-iodo-2-methyl-phenylamino) -benzoic acid; 2- (4-iodo-2-methyl-phenylamino) -4-nitro-benzoic acid; 2- (4-Bromo-2-methyl-phenylamino) -4-fluoro-benzoic acid; 2- (2-Bromo-4-iodo-phenylamino) -5-nitro-benzoic acid; 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-benzoic acid; 5-Chloro-N- (2-hydroxyethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N-methyl-benzamide; N-ethyl-4-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N, N-dimethyl-benzamide 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (1H-tetrazol-5-yl) -benzamide; 5-bromo-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-chloro-2- (4-iodo-2-methyl-phenylamino) -N, N-dimethyl-benzamide; [5-Chloro-2- (4-iodo-2-methyl-phenylamino) -benzoylamino] -acetic acid; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N-propyl-benzamide; 5-Bromo-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; N, N-diethyl-4-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 4-Fluoro-N- {3- [4- (2-hydroxy-ethyl) -piperazin-1-yl] -propyl} -2- (4-iodo-2-methyl-phenylamino) -benz amides; N, N-diethyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide; N-butyl-4-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Chloro-N, N-diethyl-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-bromo-2- (4-iodo-2-methyl-phenylamino) -N, N-dimethyl-benzamide; 5-Bromo-3,4-difluoro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; N- (2,3-dihydroxy-propyl) -3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-piperidin-1-yl-ethyl) -benzamide; 3,4-difluoro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; N- (2,3-dihydroxy-propyl) -4-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 3,4-difluoro-N- (3-hydroxy-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyrrolidin-1-yl-ethyl) -benzamide; 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyridin-4-yl-ethyl) -benzamide; 4-Fluoro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Bromo-N- (3-dimethylamino-propyl) -3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-2-phenylamino) -N- (2-morpholin-4-yl-ethyl) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-morpholin-4-yl-ethyl) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyrrolidin-1-yl-ethyl) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyridin-4-yl-ethyl) -benzamide; N- (3-Dimethylamino-propyl) -3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; N-benzyl-4-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (2-hydroxy-ethyl) -benzamide; 4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-morpholin-4-yl-ethyl) -benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-piperidin-1-yl-propyl) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-piperidin-1-yl-propyl) -benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-thiophen-2-yl-ethyl) -benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyrrolidin-1-yl-ethyl) -benzamide; 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (2-morpholin-4-yl-ethyl) -benzamide; 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N-pyridin-4-ylmethyl-benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N-pyridin-4-ylmethyl-benzamide; 2- (4-bromo-2-ylmethyl-phenylamino) -N- (3-dimethylamino-propyl) -3,4-difluoro-benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N-pyridin-4-ylmethyl-benzamide; 4-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyridin-4-yl-ethyl) -benzamide; 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (2-pyridin-4-yl-ethyl) -benzamide; 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (3-hydroxy-propyl) -benzamide; 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (2-pyrrolidin-1-yl-ethyl) -benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N-phenethyl-benzamide; 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (2-thiophen-2-yl-ethyl) -benzamide; 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N-pyridin-4-ylmethyl-benzamide; 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N-phenethyl-benzamide; 2- (4-Bromo-2-methyl-phenylamino) -3,4-difluoro-N- (2-piperidin-1-yl-ethyl) -benzamide; 5-Chloro-N- {3- [4- (2-hydroxy-ethyl) -piperazin-1-yl] -propyl} -2- (4-iodo-2-methyl-phenylamino) -benzamide ; 5-Fluoro-N- {3- [4- (2-hydroxy-ethyl) -piperazin-1-yl] -propyl} -2- (4-iodo-2-methyl-phenylamino) -benz amides; 2- (4-iodo-2-methyl-phenylamino) -5-nitro-N-pyridin-4-yl methyl-benzamide; 5-Bromo-N- {3- [4- (2-hydroxy-ethyl) -piperazin-1-yl] -propyl} -2- (4-iodo-2-methyl-phenylamino) -benz amides; 5-Chloro-N- (2-diethylamino-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-chloro-2- (4-iodo-2-methyl-phenylamino) -N- (2-piperidin-1-yl-ethyl) -benzamide; (3-hydroxy-pyrrolidin-1-yl)-[2- (4-iodo-2-methyl-phenylamino) -5-nitro-phenyl] -methanone; 5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyrrolidin-1-yl-ethyl) -benzamide; 5-Bromo-N- (2-diethylamino-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; N- {2- [bis- (2-hydroxy-ethyl) -amino] -ethyl} -5-chloro-2- (4-iodo-2-methyl-phenylamino) -benzamide; N- {2- [bis- (2-hydroxy-ethyl) -amino] -ethyl} -5-bromo-2- (4-iodo-2-methyl-phenylamino) -benzamide; N- {3- [4- (2-hydroxy-ethyl) -piperazin-1-yl] -propyl} -2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-pyridin-4-ylmethyl-benzamide; 5-Bromo-2- (4-iodo-2-ethyl-phenylamino) -N- (2-pyrrolidin-1-yl-ethyl) -benzamide; 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (2-piperidin-1-yl-ethyl) -benzamide; 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-pyrrolidin-1-yl-ethyl) -benzamide; 5-Chloro-N- (3-dimethylamino-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; N- {2- [bis- (2-hydroxy-ethyl) -amino] -ethyl} -5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Chloro-N- (3-hydroxy-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Chloro-N- (3-diethylamino-2-hydroxy-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-piperidin-1-yl-ethyl) -benzamide; 5-Bromo-N- (3-hydroxy-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (3-piperidin-1-yl-propyl) -benzamide; N- {2- [bis- (2-hydroxy-ethyl) -amino] -ethyl} -2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide; 5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N- (2-morpholin-4-yl-ethyl) -benzamide; 5-Chloro-N- (3-diethylamino-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Chloro-N- (2-diisopropylamino-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-chloro-2- (4-iodo-2-methyl-phenylamino) -N- (3-piperidin-1-yl-propyl) -benzamide; 2- (4-iodo-2-methyl-phenylamino) -5-nitro-N- (2-piperidin-1-yl-ethyl) -benzamide; 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (2-piperazin-1-yl-ethyl) -benzamide; N- (2-Diethylamino-ethyl) -5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Bromo-N- (3-dimethylamino-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; N- (3-hydroxy-propyl) -2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide; 5-Fluoro-N- (3-hydroxy-propyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; N- (3-Diethylamino-propyl) -5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; N- (3-Diethylamino-propyl) -2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide; 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (2-morpholin-4-yl-ethyl) -benzamide; 2- (4-iodo-2-methyl-phenylamino) -5-nitro-N- (3-piperidin-1-yl-propyl) -benzamide; [5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -phenyl]-(3-hydroxy-pyrrolidin-1-yl) -methanone; 5-Bromo-N- (2-diisopropylamino-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-morpholin-4-yl-ethyl) -benzamide; 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-piperidin-1-yl-propyl) -benzamide; [5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -phenyl]-[4- (2-hydroxy-ethyl) -piperazin-1-yl] -methanone; N- (3-Diethylamino-2-hydroxy-propyl) -5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; N-cyclopropyl-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Chloro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Fluoro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; N-benzyloxy-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; N-benzyloxy-5-bromo-2- (4-iodo-2-methyl-phenylamino) -benzamide; 2- (4-iodo-2-methyl-phenylamino) -5-nitro-N- (4-sulfamoyl-benzyl) -benzamide; 5-Bromo-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; N- (2-hydroxy-ethyl) -5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzamide; N- (2-hydroxy-ethyl) -2- (4-iodo-2-ethyl-phenylamino) -5-nitro-benzamide; 2- (4-iodo-2-methyl-phenylamino) -N-methyl-5-nitro-N-phenyl-benzamide; 5-Chloro-N-cyclopropyl-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-phenyl-benzamide; N-allyl-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; N-benzyloxy-5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (4-sulfamoyl-benzyl) -benzamide; N-allyl-5-chloro-2- (4-iodo-2-methyl-phenylamino) -benzamide; N-cyclopropyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide; 5-Bromo-N-cyclopropyl-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-chloro-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-phenyl-benzamide; 5-iodo-2- (4-iodo-2-methyl-phenylamino) -N- (4-sulfamoyl-benzyl) -benzamide; 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (4-sulfamoyl-benzyl) -benzamide; N-allyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide; 2- (4-iodo-2-methyl-phenylamino) -5-nitro-N- (4-sulfamoyl-benzyl) -benzamide; N-allyl-5-bromo-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-benzyl) -benzamide; N-cyclopropyl-5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-phenyl-benzamide; N-benzyloxy-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide; N-cyclohexyl-5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzamide; N-allyl-5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-iodo-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-benzyl) -benzamide; 2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-benzyl) -5-nitro-benzamide; 5-iodo-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-phenyl-benzamide; N-cyclohexyl-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Chloro-N-cyclohexyl-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-benzyl) -benzamide; 5-Bromo-N-cyclohexyl-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-chloro-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-benzyl) -benzamide; N-cyclohexyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide; N-benzyloxy-5-bromo-2- (4-iodo-2-methyl-phenylamino) -benzamide; N-benzyloxy-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Chloro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Bromo-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 2- (4-iodo-2-methyl-phenylamino) -N-methyl-5-nitro-N-phenyl-benzamide; 5-chloro-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-phenyl-benzamide; N- (2-hydroxy-ethyl) -5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Chloro-N-cyclopropyl-2- (4-iodo-2-methyl-phenylamino) -benzamide; N-allyl-5-chloro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-phenyl-benzamide; N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide; 5-Fluoro-N- (2-hydroxy-ethyl) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Bromo-N-cyclopropyl-2- (4-iodo-2-methyl-phenylamino) -benzamide; N-cyclopropyl-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (4-sulfamoyl-benzyl) -benzamide; N-cyclopropyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide; N-allyl-5-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; N-benzyloxy-5-iodo-2- (4-iodo-2-methyl-phenylamino) -benzamide; N-allyl-5-bromo-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N- (4-sulfamoyl-benzyl) -benzamide; 5-Bromo-2- (4-iodo-2-methyl-phenylamino) -N-methyl-N-phenyl-benzamide; N-allyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -benzyl alcohol; [5-Chloro-2- (4-iodo-2-methyl-phenylamino) -phenyl] -methanol; [2- (4-iodo-2-methyl-phenylamino) -5-nitro-phenyl] -methanol; [5-Bromo-2- (4-iodo-2-methyl-phenylamino) -phenyl] -methanol; And N-allyl-2- (4-iodo-2-methyl-phenylamino) -5-nitro-benzamide. The method according to claim 4, wherein the MEK inhibitor, (a) R ₁ is hydrogen, methyl, methoxy, fluorine, chlorine, or bromine; (b) R ₂ is hydrogen; (c) R ₃ , R ₄ and R ₅ are independently hydrogen, fluorine, chlorine, bromine, iodine, methyl, methoxy, or nitro; (d) R ₁₀ and R ₁₁ are independently hydrogen or methyl; (e) Z is COOR ₇ , tetrazolyl, CONR ₆ R ₇ , CONHNR ₁₀ R ₁₁ , or CH ₂ OR ₇ ; R ₆ and R ₇ are independently hydrogen, C ₁ -C ₄ alkyl, heteroaryl, or C ₃ -C ₅ cycloalkyl optionally containing one or two hetero atoms selected from O, S, or NH; R ₆ and R ₇ together with the nitrogen to which they are attached form a 5-6 membered cyclic ring optionally containing 1 or 2 additional hetero atoms selected from O, NH, or N-alkyl; Wherein said alkyl or aryl group may be unsubstituted or substituted by halo, hydroxy, methoxy, ethoxy, or heteroaryloxy; (f) Z is COOR ₇ ; (g) R ₇ is H, pentafluorophenyl, or tetrazolyl; (h) R ₃ , R ₄ and R ₅ are independently H, fluorine, or chlorine; (i) R ₄ is fluorine; (j) two of R ₃ , R ₄ and R ₅ are fluorine; Or (k) a compound of Formula (I) which is a combination of the foregoing. The method of claim 6, wherein the MEK inhibitor is Z is COOR ₇ ; R ₇ is H, pentafluorophenyl, or tetrazolyl; R ₃ and R ₅ are independently H, fluorine, or chlorine; R ₄ is fluorine; The method of claim 1, wherein the MEK inhibitor is a compound of Formula II, or a pharmaceutically acceptable salt, ester, amide or prodrug thereof. <Formula II> Wherein R _1a is hydrogen, hydroxy, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, halo, trifluoromethyl, or CN; R _2a is hydrogen; R _3a , R _4a , and R _5a are independently hydrogen, hydroxy, halo, trifluoromethyl, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, nitro, CN, or (O or NH) _m − (CH ₂ ) _n -R _9a , where R _9a is hydrogen, hydroxy, CO ₂ H or NR _10a R _11a ; n is 0-4; m is 0 or 1; R _10a and R _11a are independently hydrogen or C ₁ -C ₈ alkyl or together with the nitrogen to which they are attached 1, 2 or 3 additional hetero atoms selected from O, S, NH, or NC ₁ -C ₈ alkyl Optionally contain 3- to 10-membered cyclic rings; R _6a is hydrogen, C ₁ -C ₈ alkyl, (CO) -C ₁ -C ₈ alkyl, aryl, aralkyl, or C ₃ -C ₁₀ cycloalkyl; R _7a optionally represents a hetero atom selected from hydrogen, C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C ₃ -C ₁₀ (cycloalkyl or O, S, or NR _9a Containing cycloalkyl); Wherein the alkyl, alkenyl, aryl, heteroaryl, heterocyclic and alkynyl groups are unsubstituted or halo, hydroxy, C ₁ -C ₆ alkoxy, amino, nitro, C ₁ -C ₄ alkylamino, di ( C ₁ -C ₄ ) alkylamino, C ₃ -C ₆ cycloalkyl, phenyl, phenoxy, C ₃ -C ₅ heteroaryl or heterocyclic radicals, or C ₃ -C ₅ heteroaryloxy or heterocyclic radicals- May be substituted by oxy; Or R _6a and R _7a together with N to which they are attached will form a 5- to 10-membered cyclic ring optionally containing 1, 2 or 3 additional hetero atoms selected from O, S, or NR _10a R _11a Can be. The method according to claim 8, wherein the MEK inhibitor, (a) R _1a is H, methyl, fluorine, or chlorine; (b) R _2a is H; R _3a , R _4a , and R _5a are each H, Cl, nitro, or F; (c) R _6a is H; (d) R _7a is methyl, ethyl, 2-propenyl, propyl, butyl, pentyl, hexyl, cyclopropylmethyl, cyclobutylmethyl, cyclopropylmethyl, or cyclopropylethyl; (e) The 4 'position is a compound having a structure of Formula II wherein I is more than Br. 10. The method of claim 9, wherein the MEK inhibitor is F, wherein R _4a is at position 4, para for CO-NR _6a -OR _7a group and meta for crosslinked nitrogen; At least one R _3a and R _5a is F or Cl; R _1a is a compound having a structure of Formula II wherein methyl is chlorine. The method according to claim 8, wherein the MEK inhibitor, 4-fluoro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (methoxy) -benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (prop-2-ynyloxy) -benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-phenoxyethoxy) -benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-thienylmethoxy) -benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (prop-2-enyloxy) -benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (cyclopropylmethoxy) -benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (cyclopentoxy) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-furylmethoxy) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N-ethoxy-benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (but-2-enyloxy) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (cyclopropylmethoxy) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (1-methylprop-2-ynyloxy) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-phenylprop-2-ynyloxy) -benzamide; 3,4-Difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-5-phenylpent-2-ene-4-ynyloxy) -benzamide 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (prop-2-ynyloxy) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (propoxy) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (cyclobutyloxy) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-thienylmethoxy) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-methyl-prop-2-enyloxy) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (2-phenoxyethoxy) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (but-2-enyloxy) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (but-3-ynyloxy) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (cyclopentyloxy) -benzamide; 3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3- (2-fluorophenyl) -prop-2-ynyloxy) -benzamide; 5-Bromo-3,4-difluoro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (n-propoxy) -benzamide; 5-Bromo-3,4-difluoro-N- (furan-3-ylmethoxy) -2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-bromo-N- (but-2-enyloxy) -3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-bromo-N-butoxy-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-but-2-enyloxy) -benzamide; 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3-methyl-pent-2-en-4-ynyloxy) -benzamide ; 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-benzyl) -N- [5- (3-methoxy-phenyl) -3-methyl-pent-2- En-4-ynyloxy] -benzamide; 5-bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (prop-2-ynyloxy) -benzamide; 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- [3- (3-methoxy-phenyl) -prop-2-ynyloxy ] -Benzamide; 5-bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (thiophen-2-ylmethoxy) -benzamide; 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (pyridin-3-ylmethoxy) -benzamide; 5-Bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (3- (2-fluorophenyl) -prop-2-ynyloxy) Benzamide; 5-bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (ethoxy) -benzamide; 5-bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (cyclopropylmethoxy) -benzamide; 5-bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (isopropoxy) -benzamide; 5-bromo-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -N- (but-3-ynyloxy) -benzamide; 5-Chloro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Chloro-2- (4-iodo-2-methyl-phenylamino) -N- (tetrahydro-pyran-2-yloxy) -benzamide; 5-chloro-2- (4-iodo-2-methyl-phenylamino) -N-methoxy-benzamide; 4-bromo-2- (4-iodo-2-methyl-phenylamino) -N-phenylmethoxy-benzamide; 4-fluoro-2- (4-iodo-2-methyl-phenylamino) -N-phenylmethoxy-benzamide; 5-Fluoro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-iodo-2- (4-iodo-2-methyl-phenylamino) -N-phenylmethoxy-benzamide; 5-fluoro-2- (4-iodo-2-methyl-phenylamino) -N- (tetrahydropyran-2-yloxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (3-phenylprop-2-ynyloxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (3-furylmethoxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (2-thienylmethoxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (but-3-ynyloxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (2-methyl-prop-2-enyloxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (but-2-enyloxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (methoxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (ethoxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (cyclobutoxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (isopropoxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (2-phenoxyethoxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (cyclopropylmethoxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (n-propoxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (1-methyl-prop-2-ynyloxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (3- (3-fluorophenyl) -prop-2-ynyloxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (4,4-dimethylpent-2-ynyloxy) -benzamide; 3,4-difluoro-2- (4-bromo-2-methyl-phenylamino) -N- (cyclopentoxy) -benzamide; 3,4,5-trifluoro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Chloro-3,4-difluoro-N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-bromo-3,4-difluoro-2- (2-fluoro-4-iodo-phenylamino) -N-hydroxy-benzamide; N-hydroxy-2- (4-iodo-2-methyl-phenylamino) -4-nitro-benzamide; 3,4,5-trifluoro-2- (2-fluoro-4-iodo-phenylamino) -N-hydroxy-benzamide; 5-Chloro-3,4-difluoro-2- (2-fluoro-4-iodo-phenylamino) -N-hydroxy-benzamide; 5-bromo-2- (2-chloro-4-iodo-phenylamino) -3,4-difluoro-N-hydroxy-benzamide; 2- (2-fluoro-4-iodo-phenylamino) -N-hydroxy-4-nitro-benzamide; 2- (2-Chloro-4-iodo-phenylamino) -3,4,5-trifluoro-N-hydroxy-benzamide; 5-chloro-2- (2-chloro-4-iodo-phenylamino) -3,4-difluoro-N-hydroxy-benzamide; 5-bromo-2- (2-bromo-4-iodo-phenylamino) -3,4-difluoro-N-hydroxy-benzamide; 2- (2-Chloro-4-iodo-phenylamino) -N-hydroxy-4-methyl-benzamide; 2- (2-bromo-4-iodo-phenylamino) -3,4,5-trifluoro-N-hydroxy-benzamide; 2- (2-Bromo-4-iodo-phenylamino) -5-chloro-3,4-difluoro-N-hydroxy-benzamide; 2- (2-Bromo-4-iodo-phenylamino) -N-hydroxy-4-nitro-benzamide; 4-fluoro-2- (2-fluoro-4-iodo-phenylamino) -N-hydroxy-benzamide; 3,4-difluoro-2- (2-fluoro-4-iodo-phenylamino) -N-hydroxy-benzamide; 2- (2-Chloro-4-iodo-phenylamino) -4-fluoro-N-hydroxy-benzamide; 2- (2-Chloro-4-iodo-phenylamino) -3,4-difluoro-N-hydroxy-benzamide; 2- (2-Bromo-4-iodo-phenylamino) -4-fluoro-N-hydroxy-benzamide; 2- (2-bromo-4-iodo-phenylamino) -3,4-difluoro-N-hydroxy-benzamide; N-cyclopropylmethoxy-3,4,5-trifluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-Chloro-N-cyclopropylmethoxy-3,4-difluoro-2- (4-iodo-2-methyl-phenylamino) -benzamide; 5-bromo-N-cyclopropylmethoxy-3,4-difluoro-2- (2-fluoro-4-iodo-phenylamino) -benzamide; N-cyclopropylmethoxy-2- (4-iodo-2-methyl-phenylamino) -4-nitro-benzamide; N-cyclopropylmethoxy-3,4,5-trifluoro-2- (2-fluoro-4-iodo-phenylamino) -benzamide; 5-Chloro-N-cyclopropylmethoxy-3,4-difluoro-2- (2-fluoro-4-iodo-phenylamino) -benzamide; 5-bromo-2- (2-chloro-4-iodo-phenylamino) -N-cyclopropylmethoxy-3,4-difluoro-benzamide; N-cyclopropylmethoxy-2- (2-fluoro-4-iodo-phenylamino) -4-nitro-benzamide; 2- (2-Chloro-4-iodo-phenylamino) -N-cyclopropylmethoxy-3,4,5-trifluoro-benzamide; 5-chloro-2- (2-chloro-4-iodo-phenylamino) -N-cyclopropylmethoxy-3,4-difluoro-benzamide; 5-bromo-2- (2-bromo-4-iodo-phenylamino) -N-ethoxy-3,4-difluoro-benzamide; 2- (2-Chloro-4-iodo-phenylamino) -N-ethoxy-4-nitro-benzamide; 2- (2-bromo-4-iodo-phenylamino) -N-cyclopropylmethoxy-3,4,5-trifluoro-benzamide; 2- (2-Bromo-4-iodo-phenylamino) -5-chloro-N-cyclopropylmethoxy-3,4-difluoro-benzamide; 2- (2-Bromo-4-iodo-phenylamino) -N-cyclopropylmethoxy-4-nitro-benzamide; N-cyclopropylmethoxy-4-fluoro-2- (2-fluoro-4-iodo-phenylamino) -benzamide; N-cyclopropylmethoxy-3,4-difluoro-2- (2-fluoro-4-iodo-phenylamino) -benzamide; 2- (2-Chloro-4-iodo-phenylamino) -N-cyclopropylmethoxy-4-fluoro-benzamide; 2- (2-Chloro-4-iodo-phenylamino) -N-cyclopropylmethoxy-3,4-difluoro-benzamide; 2- (2-Bromo-4-iodo-phenylamino) -N-cyclopropylmethoxy-4-fluoro-benzamide; And A compound having a formula selected from 2- (2-bromo-4-iodo-phenylamino) -N-cyclopropylmethoxy-3,4-difluoro-benzamide. The method of claim 1, wherein the MEK inhibitor is selected from the group consisting of 2- (2-chloro-4-iodophenylamino) -5-chloro-N-cyclopropylmethoxy-3,4-difluorobenzamide (PD297189); 2- (4-iodophenylamino) -N-cyclopropylmethoxy-5-chloro-3,4-difluorobenzamide (PD 297190); 2- (4-iodophenylamino) -5-chloro-3,4-difluorobenzoic acid (PD 296711); 2- (2-Chloro-4-iodophenylamino) -5-chloro-3,4-difluorobenzoic acid (PD 296770); 5-Chloro-3,4-difluoro-2- (4-iodo-2-methylphenylamino) -benzoic acid (PD 296767); And 5-chloro-N-cyclopropylmethoxy-3,4-difluoro-2- (4-iodo-2-methylphenylamino) -benzamide (PD 298127). To a patient who has or will receive a transplant, 2- (2-chloro-4-iodophenylamino) -N-cyclopropylmethoxy-3,4-difluorobenzamide (PD184352); 2- (2-methyl-4-iodophenylamino) -N-hydroxy-4-fluorobenzamide (PD170611); 2- (2-methyl-4-iodophenylamino) -N-hydroxy-3,4-difluoro-5-bromobenzamide (PD171984); 2- (2-methyl-4-iodophenylamino) -N-cyclopropylmethoxy-3,4-difluoro-5-bromobenzamide (PD177168); 2- (2-methyl-4-iodophenylamino) -N-cyclobutylmethoxy-3,4-difluoro-5-bromobenzamide (PD180841); 2- (2-chloro-4-iodophenylamino) -N-cyclopropylmethoxy-3,4-difluoro-5-bromobenzamide (PD 184161); 2- (2-chloro-4-iodophenylamino) -N-hydroxy-3,4-difluoro-5-bromobenzamide (PD184386); 2- (2-chloro-4-iodophenylamino) -N-cyclobutylmethoxy-3,4-difluorobenzamide (PD185625); 2- (2-chloro-4-iodophenylamino) -N-hydroxy-4-fluorobenzamide (PD185848); 2- (2-methyl-4-iodophenylamino) -N-hydroxy-3,4-difluorobenzamide (PD188563); 2- (2-methyl-4-iodophenylamino) -N-cyclopropylmethoxy-3,4,5-trifluorobenzamide (PD198306); And Administering an immunosuppressive effective amount of a compound selected from 2- (2-chloro-4-iodophenylamino) -N-cyclopropylmethoxy-4-fluorobenzamide (PD 203311) A method for preventing and inhibiting rejection of transplanted organs, cells, tissues, or limbs. Administering an effective amount of 2- (2-chloro-4-iodophenylamino) -N-cyclopropylmethoxy-3,4-difluorobenzamide to a patient in need of prevention or maintenance of transplant tissue rejection A method for preventing or maintaining transplant tissue rejection in a mammal. Administering an effective amount of 2- (2-methyl-4-iodophenylamino) -N-cyclopropylmethoxy-3,4,5-trifluorobenzamide to patients in need of prevention or maintenance of graft rejection A method for preventing or maintaining transplant tissue rejection in a mammal.